Processes for producing a fermentation product

ABSTRACT

The present invention relates to polypeptides having trehalase activity, particularly derived from  Talaromyces . The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides for the production of ethanol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofinternational application no. PCT/US2018/039443 filed Jun. 26, 2018,which claims priority or the benefit under 35 U.S.C. 119 of U.S.provisional application No. 62/526,133 filed Jun. 28, 2017, the contentsof which are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides having treahalase activityand polynucleotides encoding the polypeptides. The invention alsorelates to nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides as well as methods of producing the polypeptides.The invention also relates to processes of producing fermentationproducts using a trehalase of the invention.

Description of the Related Art

Trehalose is a stable disaccharide sugar consisting of two sugarmonomers (glucose). Trehalose is accumulated in yeast as a response tostress in up to 10-15% of cell dry weight (GrBa et al. (1975) Eur. J.Appl. Microbiol. 2:29-37). Trehalose cannot be metabolized by the yeast.The enzyme trehalase cleaves trehalose into two glucose units.

Trehalases are classified in EC 3.2.1.28 (alpha,alpha-trehalase) and EC.3.2.1.93 (alpha,alpha-phosphotrehalase). The EC classes are based onrecommendations of the Nomenclature Committee of the International Unionof Biochemistry and Molecular Biology (IUBMB). Description of EC classescan be found on the internet, e.g., on the expasy website. The twoenzyme classes are both referred to as “trehalases”. Examples of neutraltrehalases include treahalases from Saccharomyces cerevisiae(Londesborouh et al. (1984) Characterization of two trehalases frombaker's yeast” Biochem J 219, 511-518; Mucor roxii (Dewerchin et al(1984), “Trehalase activity and cyclic AMP content during earlydevelopment of Mucor rouxii spores”, J. Bacteriol. 158, 575-579);Phycomyces blakesleeanus (Thevelein et al (1983), “Glucose-inducedtrehalase activation and trehalose mobilization during early germinationof Phycomyces blakesleeanus spores” J. Gen Microbiol. 129, 719-726);Fusarium oxysporium (Amaral et al (1996), “Comparative study of twotrehalase activities from Fusarium oxysporium var Linii” Can. JMicrobiol. 41, 1057-1062). Examples of neutral trehalases include, butare not limited to, trehalases from Saccharomyces cerevisiae (Parvaeh etal. (1996) Purification and biochemical characterization of the ATH1gene product, vacuolar acid trehalase from Saccharomyces cerevisae” FEBSLett. 391, 273-278); Neorospora crassa (Hecker et al (1973), “Locationof trehalase in the ascospores of Neurospora: Relation to ascosporedormancy and germination”. J. Bacteriol. 115, 592-599); Chaetomiumaureum (Sumida et al. (1989), “Purification and some properties oftrehalase from Chaetomium aureum MS-27. J. Ferment. Bioeng. 67, 83-86);Aspergillus nidulans (d'Enfert et al. (1997), “Molecularcharacterization of the Aspergillus nidulans treA gene encoding an acidtrehalase required for growth on trehalose. Mol. Microbiol. 24,203-216); Humicola grisea (Zimmermann et al. (1990).” Purification andproperties of an extracellular conidial trehalase from Humicola griseavar. thermoidea”, Biochim. Acta 1036, 41-46); Humicola grisea (Cardelloet al. (1994), “A cytosolic trehalase from the thermophilhilic fungusHumicola grisea var. thermoidea’, Microbiology UK 140, 1671-1677;Scytalidium thermophilum (Kadowaki et al. (1996), “Characterization ofthe trehalose system from the thermophilic fungus Scytalidiumthermophilum” Biochim. Biophys. Acta 1291, 199-205); and Fusariumoxysporium (Amaral et al (1996), “Comparative study of two trehalaseactivities from Fusarium oxysporium var Linii” Can. J Microbiol. 41,1057-1062).

A trehalase is also know from soybean (Aeschbachet et al (1999)”Purification of the trehalase GmTRE1 from soybean nodules and cloning ofits cDNA”, Plant Physiol 119, 489-496).

Trehalases are also present in small intestine and kidney of mammals.

WO 2009/121058 (Novozymes) concerns a method of fermenting sugarsderived from plant material into a fermentation product, such asethanol, using a fermenting organism by adding one or more trehalaseinto in the fermentation medium.

WO 2012/027374 (Dyadic) discloses a trehalase from Myceliophthorathermophila which can be used in an enzyme mixture for degradinglignocellulosic biomass to fermentable sugars.

WO 2013/148993 (Novozymes) discloses a process of producing afermentation product, such as ethanol, from starch-containing materialby liquefying, saccharifying and fermenting the starch-containingmaterial wherein wherein a carbohydrate-source generating enzyme, acellulolytic composition and a trehalase is present in fermentation. Atrehalase from Trichoderma reesei is disclosed.

WO 2015/065978 (Danisco US Inc.) discloses a method of increasing theproduction of ethanol from a liquefact in a fermentation reactionincluding fermenting the liquefact with a glucoamylase, a fermentingorganism and a trehalase and recovering the ethanol and otherfermentation products at the end of the fermentation.

WO 2016/205127 (Novozymes) discloses a trehalase from Myceliophthorasepedonium belonging to Family 37 Glucoside Hydrolases (“GH37”) asdefined by CAZY (available online), having high thermostability and abroad pH range. It was also found that an increased ethanol yield can beobtained when adding a trehalase to fermentation in an ethanol process.

Fujii T., et al., 2014, Taxonomic revision of the cellulose-degradingfungus Acremonium cellulolyticus nomen nudum to Talaromyces based onphylogenetic analysis. FEMS Microbiology Letters, 351: 32-41 andUniprot:AOAOB8MYG3 disclose trehalases from Talaromyces cellulyticus,and Uniprot:AOA1 L9RM22 discloses a trehalase from Aspergillus wentii.

A trehalase from Talaromyces verruculosus was published in 2015 as partof a genome sequence on the NCBI website as assembly GCA 001305275.1;(polypeptide identified as EFP5BRM8N).

There is still a need for providing enzymes or enzyme compositionsuitable for use in processes for producing fermentation products, suchas ethanol, in increased yields.

SUMMARY OF THE INVENTION

The present invention provides polypeptides having trehalase activityand polynucleotides encoding the polypeptides. The trehalases accordingto the invention have good stability towards degradation by proteasesand high thermo-stability. The trehalases are preferably obtained from afungus of the genus Talaromyces.

Accordingly, the present invention relates to polypeptides havingtrehalase activity selected from the group consisting of:

(a) a polypeptide having at least 93% sequence identity to the maturepolypeptide of SEQ ID NO: 21 or at least 70% sequence identity to themature polypeptide of SEQ ID NO: 23;

(b) a polypeptide encoded by a polynucleotide having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 20 or the cDNA sequence thereof; or at least 80% sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 22 or the cDNAsequence thereof;

(c) a variant of the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO:23 comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions; and

(d) a fragment of the polypeptide of (a), (b), or (c), that hastrehalase activity.

In further aspect the present invention relates to polynucleotidesencoding the variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.In a further aspect the present invention relates to compositionscomprising the variants of the invention.

The present invention also relates to a process of producing afermentation product, comprising

(a) liquefying a starch-containing material with an alpha-amylase;

optionally pre-saccharifying the liquefied material before step (b);

(b) saccharifying the liquefied material;

(c) fermenting using a fermentation organism;

wherein

-   -   i) a glucoamylase;    -   ii) a trehalase of the invention;    -   iii) optionally a cellulolytic enzyme composition and/or a        protease; are present and/or added during

saccharification step (b);

fermentation step (c);

simultaneous saccharification and fermentation;

optionally presaccharification step before step (b).

In still further aspects the present invention relates to process ofproducing fermentation products from starch-containing materialcomprising:

(i) saccharifying a starch-containing material at a temperature belowthe initial gelatinization temperature; and

(ii) fermenting using a fermentation organism;

wherein saccharification and/or fermentation is done in the presence ofthe following enzymes: glucoamylase, alpha-amylase, trehalase of any ofclaims 1-7, and optionally a protease and/or a cellulolytic enzymecomposition.

Definitions

Trehalase: The term “trehalase” means an enzyme which degrades trehaloseinto its unit monosaccharides (i.e., glucose). Trehalases are classifiedin EC 3.2.1.28 (alpha,alpha-trehalase) and EC. 3.2.1.93(alpha,alpha-phosphotrehalase). The EC classes are based onrecommendations of the Nomenclature Committee of the International Unionof Biochemistry and Molecular Biology (IUBMB). Description of EC classescan be found on the internet, e.g., on the expasy website. Trehalasesare enzymes that catalyze the following reactions:

EC 3.2.1.28:Alpha,alpha-trehalose+H₂O⇔2 D-glucose;

EC 3.2.1.93:Alpha,alpha-trehalose 6-phosphate+H₂O⇔D-glucose+D-glucose 6-phosphate.

For purposes of the present invention, trehalase activity may bedetermined according to “Trehalase Assay” procedure described in the“Materials & Methods”-section. In one aspect, the polypeptides of thepresent invention have at least 20%, e.g., at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 100% of the trehalase activity of the mature polypeptide of SEQID NO: 21. In another aspect, the polypeptides of the present inventionhave at least 20%, e.g., at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 100% ofthe trehalase activity of the mature polypeptide of SEQ ID NO: 23. In apreferred embodiment a trehalase of the invention is a Family 65Glycoside Hydrolase (“GH65 trehalase”).

In one embodiment the trehalases according to the invention, SEQ ID NO:21 and/or SEQ ID NO: 23 have a denaturing temperature Td (measured bythe TSA assay) of at least 60° C., at least 61° C., at least 62° C., atleast 63° C., at least 64° C., at least 65° C., at least 66° C., atleast 67° C., such as at least 68° C.

In another embodiment the trehalases according to the invention, SEQ IDNO: 21 and/or SEQ ID NO: 23 have a residual activity after 3 daysincubation at 40° C. with an A. niger protease mixture of 100%.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme. In oneembodiment the catalytic domain is amino acids 387 to 769 of SEQ ID NO:21. In another embodiment the catalytic domain is amino acids 384 to 799of SEQ ID NO: 23.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has trehalaseactivity. In one aspect, a fragment contains amino acids 387 to 769 ofSEQ ID NO: 21. In one aspect, a fragment contains at least amino acids384 to 799 of SEQ ID NO: 23.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to thermostability,wherein the denaturing temperature (Td) measure by Thermal Shift Assay(TSA) is at least 60° C., at least 61° C., at least 62° C., at least 63°C., at least 64° C., at least 65° C., at least 66° C., at least 67° C.,such as at least 68° C. and stability against protease degradation, inparticular degradation by Aspergillus niger protease mixture.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 19 to 1038 of SEQ ID NO: 21. In anotheraspect, the mature polypeptide is amino acids 21 to 1089 of SEQ ID NO:23. It is known in the art that a host cell may produce a mixture of twoof more different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving trehalase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 55 to 2037, and 2085 to 3161 of SEQ ID NO: 20.Nucleotides 1 to 54 of SEQ ID NO: 20 encode a signal peptide. In anotheraspect, the mature polypeptide coding sequence is nucleotides 61 to1740, and 2026 to 2313, and 2367 to 3605 of SEQ ID NO: 22. Nucleotides 1to 60 of SEQ ID NO: 22 encode a signal peptide.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences. In one embodiment the one or more control sequences areheterologous (of different origin/species) to the coding sequenceencoding the polypeptide of the invention.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent trehalse: The term “parent” or “parent trehalase” meansany polypeptide with trehalase activity to which an alteration is madeto produce an enzyme variants of the present invention.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stringency conditions: The term “high stringency conditions” means forprobes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for minutes using 2×SSC, 0.2% SDS at 70° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having trehalase activity. In one aspect, a subsequencecontains at least nucleotides 1159 to 2037, and 2085 to 2354 of SEQ IDNO: 20. In one aspect, a subsequence contains at least nucleotides 1150to 1740, and 2026 to 2313, and 2367 to 2735 of SEQ ID NO: 22.

Variant: The term “variant” means a polypeptide having trehalaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition The variants of the present invention have at least 20%, e.g.,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 100% of the trehalase activity ofthe polypeptide of SEQ ID NO: 21. The variants of the present inventionhave at least 20%, e.g., at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 100% ofthe trehalase activity of the polypeptide of SEQ ID NO: 23.

Wild-type trehalase: The term “wild-type” trehalase means a trehalaseexpressed by a naturally occurring microorganism, such as a bacterium,yeast, or filamentous fungus found in nature.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Trehalse Activity

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 21 of at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%, which have trehalase activity. In oneaspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 21.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 21 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, andwherein the polypeptide has at least at least 70% of the trehalaseactivity of the mature polypeptide of SEQ ID NO: 21, and wherein thedenaturing temperature measured by TSA is at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 21 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 75% of the trehalase activity of the mature polypeptideof SEQ ID NO: 21, and wherein the denaturing temperature measured by TSAis at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 21 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 80% of the trehalase activity of the mature polypeptideof SEQ ID NO: 21, and wherein the denaturing temperature measured by TSAis at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 21 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 85% of the trehalase activity of the mature polypeptideof SEQ ID NO: 21, and wherein the denaturing temperature measured by TSAis at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 21 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 90% of the trehalase activity of the mature polypeptideof SEQ ID NO: 21, and wherein the denaturing temperature measured by TSAis at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 21 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 95% of the trehalase activity of the mature polypeptideof SEQ ID NO: 21, and wherein the denaturing temperature measured by TSAis at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 21 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 100% of the trehalase activity of the mature polypeptideof SEQ ID NO: 21, and wherein the denaturing temperature measured by TSAis at least 60° C.

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 23 of at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which havetrehalase activity. In one aspect, the polypeptides differ by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide of SEQ ID NO: 23.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 23 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, andwherein the polypeptide has at least at least 70% of the trehalaseactivity of the mature polypeptide of SEQ ID NO: 23, and wherein thedenaturing temperature measured by TSA is at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 23 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein thepolypeptide has at least at least 75% of the trehalase activity of themature polypeptide of SEQ ID NO: 23, and wherein the denaturingtemperature measured by TSA is at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 23 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, andwherein the polypeptide has at least at least 80% of the trehalaseactivity of the mature polypeptide of SEQ ID NO: 23, and wherein thedenaturing temperature measured by TSA is at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 23 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, andwherein the polypeptide has at least at least 85% of the trehalaseactivity of the mature polypeptide of SEQ ID NO: 23, and wherein thedenaturing temperature measured by TSA is at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 23 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, andwherein the polypeptide has at least at least 90% of the trehalaseactivity of the mature polypeptide of SEQ ID NO: 23, and wherein thedenaturing temperature measured by TSA is at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 23 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, and wherein thepolypeptide has at least at least 95% of the trehalase activity of themature polypeptide of SEQ ID NO: 23, and wherein the denaturingtemperature measured by TSA is at least 60° C.

In a particular embodiment the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 23 of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, andwherein the polypeptide has at least at least 100% of the trehalaseactivity of the mature polypeptide of SEQ ID NO: 23, and wherein thedenaturing temperature measured by TSA is at least 60° C.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO: 21 or an allelic variant thereof; or is afragment thereof having trehalase activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 21. In another aspect, the polypeptide comprises or consists ofamino acids 19 to 1038 of SEQ ID NO: 21.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO: 23 or an allelic variant thereof; or is afragment thereof having trehalase activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 23. In another aspect, the polypeptide comprises or consists ofamino acids 21 to 1089 of SEQ ID NO: 23.

In another embodiment, the present invention relates to a polypeptidehaving trehalase activity encoded by a polynucleotide that hybridizesunder very low stringency conditions, low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 20, (ii) the cDNAsequence thereof, or (iii) the full-length complement of (i) or (ii)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.). In an embodiment, the polypeptidehas been isolated.

In another embodiment, the present invention relates to a polypeptidehaving trehalase activity encoded by a polynucleotide that hybridizesunder very low stringency conditions, low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 22, (ii) the cDNAsequence thereof, or (iii) the full-length complement of (i) or (ii)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.). In an embodiment, the polypeptidehas been isolated.

The polynucleotide of SEQ ID NO: 20 or SEQ ID NO: 22 or a subsequencethereof, as well as the polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23 ora fragments thereof may be used to design nucleic acid probes toidentify and clone DNA encoding polypeptides having trehalase activityfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having trehalase activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequencethereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 20 or 22; (ii) the mature polypeptide coding sequenceof SEQ ID NO: 20 or 22; (iii) the cDNA sequence thereof; (iv) thefull-length complement thereof; or (v) a subsequence thereof; under verylow to very high stringency conditions. Molecules to which the nucleicacid probe hybridizes under these conditions can be detected using, forexample, X-ray film or any other detection means known in the art.

In another embodiment, the present invention relates to an polypeptidehaving trehalase activity encoded by a polynucleotide having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 20 orthe cDNA sequence thereof of at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%. In afurther embodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to an polypeptidehaving trehalase activity encoded by a polynucleotide having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 22 orthe cDNA sequence thereof of at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%. In afurther embodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 21 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 21 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 23 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 23 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant moleculesare tested for trehalase activity to identify amino acid residues thatare critical to the activity of the molecule. See also, Hilton et al.,1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme orother biological interaction can also be determined by physical analysisof structure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identity of essential amino acids can also beinferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Trehalase Activity

A polypeptide having trehalase activity of the present invention may beobtained from microorganisms of genus Talaromyces. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

In one aspect, the polypeptide is a Talaromyces polypeptide, e.g., apolypeptide obtained from Talaromyces funiculosus or from Talaromycesleycettanus, such as e.g., CBS 398.68.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Catalytic Domains

In one embodiment, the present invention also relates to catalyticdomains having a sequence identity to amino acids 387 to 769 of SEQ IDNO: 21 of at least at least 80%, at least 85%, at least 90, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%. In one aspect, the catalyticdomains comprise amino acid sequences that differ by up to 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 387 to769 of SEQ ID NO: 21.

The catalytic domain preferably comprises or consists of amino acids 387to 769 of SEQ ID NO: 21; or is a fragment thereof having trehalaseactivity.

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 1159 to 2037, and 2085 to 2354 of SEQ ID NO: 20 or the cDNAsequence thereof of at least 80%, at least 85%, at least 90, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%.

The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 1159 to 2037, and 2085 to 2354 of SEQ ID NO: 20.

In another embodiment, the present invention also relates to catalyticdomain variants of amino acids 387 to 769 of SEQ ID NO: 21 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids387 to 769 of SEQ ID NO: 21 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9,or 10.

In one embodiment, the present invention also relates to catalyticdomains having a sequence identity to amino acids 384 to 799 of SEQ IDNO: 23 of at least 75%, at least 80%, at least 85%, at least 90, atleast at least 93%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the catalytic domainscomprise amino acid sequences that differ by up to 10 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 384 to 799 of SEQ IDNO: 23.

The catalytic domain preferably comprises or consists of amino acids 384to 799 of SEQ ID NO: 23 or an allelic variant thereof; or is a fragmentthereof having trehalase activity.

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 1150 to 1740, and 2026 to 2313, and 2367 to 2735 of SEQ IDNO: 22 or the cDNA sequence thereof of at least 75%, at least 80%, atleast 85%, at least 90, at least 93%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%.

The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 1150 to 1740, and 2026 to 2313, and 2367 to 2735of SEQ ID NO: 22.

In another embodiment, the present invention also relates to catalyticdomain variants of amino acids 384 to 799 of SEQ ID NO: 23 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids384 to 799 of SEQ ID NO: 23 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9,or 10.

Polynucleotides

The present invention also relates to polynucleotides encoding apolypeptide, or a catalytic domain of the present invention, asdescribed herein. In an embodiment, the polynucleotide encoding thepolypeptide, or catalytic domain, of the present invention has beenisolated.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Talaromyces, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences. In one particular embodiment at least one control sequence isheterologous (of different origin/species) to the polynucleotideencoding the variant of the present invention.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including variant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and variant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase Ill,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites.

Alternatively, the polynucleotide may be expressed by inserting thepolynucleotide or a nucleic acid construct comprising the polynucleotideinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Suitable markers for yeast host cells include, but are not limited to,ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for usein a filamentous fungal host cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. In a particular embodiment the recombinant host cellcomprises the polynucleotide encoding a trehalase polypeptide of thepresent invention in which the said polynucleotide is heterologous (ofdifferent origin/species) to the host cell. A construct or vectorcomprising a polynucleotide is introduced into a host cell so that theconstruct or vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.In a particular embodiment the whole broth formulation is generated byfermentation of a recombinant host cell of the invention. Thefermentation broth product further comprises additional ingredients usedin the fermentation process, such as, for example, cells (including, thehost cells containing the gene encoding the polypeptide of the presentinvention which are used to produce the polypeptide of interest), celldebris, biomass, fermentation media and/or fermentation products. Insome embodiments, the composition is a cell-killed whole brothcontaining organic acid(s), killed cells and/or cell debris, and culturemedium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis. In some embodiments, the cell-killed whole broth orcomposition contains the spent cell culture medium, extracellularenzymes, and killed filamentous fungal cells. In some embodiments, themicrobial cells present in the cell-killed whole broth or compositioncan be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide having trehalase activity of the present invention.Preferably, the compositions are enriched in such a polypeptide. Theterm “enriched” indicates that the trehalase activity of the compositionhas been increased, e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of hydrolase, isomerase, ligase, lyase,oxidoreductase, or transferase, e.g., an alpha-galactosidase,alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase,beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, glucoamylase, invertase, laccase, lipase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

In an embodiment the composition comprises a trehalase of the inventionand a glucoamylase. In an embodiment the composition comprises atrehalase of the invention and a glucoamylase derived from Talaromycesemersonii (e.g., SEQ ID NO: 4). In an embodiment the compositioncomprises a trehalase of the invention and a glucoamylase derived fromGloeophyllum, such as G. serpiarium (e.g., SEQ ID NO: 5) or G. trabeum(e.g., SEQ ID NO: 6). In an embodiment the composition comprises atrehalase of the invention, a glucoamylase and an alpha-amylase. In anembodiment the composition comprises a trehalase of the invention, aglucoamylase and an alpha-amylase derived from Rhizomucor, preferably astrain the Rhizomucor pusillus, such as a Rhizomucor pusillusalpha-amylase hybrid having an linker (e.g., from Aspergillus niger) andstarch-bonding domain (e.g., from Aspergillus niger). In an embodimentthe composition comprises a trehalase of the invention, a glucoamylase,an alpha-amylase and a cellulolytic enzyme composition. In an embodimentthe composition comprises a trehalase of the invention, a glucoamylase,an alpha-amylase and a cellulolytic enzyme composition, wherein thecellulolytic composition is derived from Trichoderma reesei. In anembodiment the composition comprises a trehalase of the invention, aglucoamylase, an alpha-amylase and a protease. In an embodiment thecomposition comprises a trehalase of the invention, a glucoamylase, analpha-amylase and a protease. The protease may be derived fromThermoascus aurantiacus. In an embodiment the composition comprises atrehalase of the invention, a glucoamylase, an alpha-amylase, acellulolytic enzyme composition and a protease. In an embodiment thecomposition comprises a trehalase of the invention, a glucoamylase,e.g., derived from Talaromyces emersonii, Gloeophyllum serpiarium orGloephyllum trabeum, an alpha-amylase, e.g., derived from Rhizomucorpusillus, in particular one having a linker and starch-binding domain,in particular derived from Aspergillus niger, in particular one havingthe following substitutions: G128D+D143N (using SEQ ID NO: 7 fornumbering); a cellulolytic enzyme composition derived from Trichodermareesei, and a protease, e.g., derived from Thermoascus aurantiacus orMeripilus giganteus.

Examples of specifically contemplated secondary enzymes, e.g., aglucoamylase from Talaromyces emersonii shown in SEQ ID NO: 4 herein ora glucoamylase having, e.g., at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO: 4 herein can be found in the “Enzymes”section below. The compositions may be prepared in accordance withmethods known in the art and may be in the form of a liquid or a drycomposition. The compositions may be stabilized in accordance withmethods known in the art.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

Processes of the Invention

Producing a Fermentation Product from Gelatinized Starch Material Usinga Trehalase of the Invention

In this aspect the present invention relates to producing a fermentationproduct, in particular ethanol, from gelatinized and/or ungelatinizedstarch-containing material or cellulosic material. Fermentable sugarsgenerated during saccharification/hydrolysis are converted to thedesired fermentation in question, in particular ethanol, duringfermentation by a fermenting organism, in particular yeast.

In an embodiment the invention relates to processes of producing afermentation product, in particular ethanol, comprising

(a) liquefying a starch-containing material with an alpha-amylase;

optionally pre-saccharifying the liquefied material before step (b);

(b) saccharifying the liquefied material;

(c) fermenting using a fermentation organism;

wherein

i) a glucoamylase;

ii) a trehalase of the invention;

iii) optionally a cellulolytic enzyme composition and/or a protease;

are present and/or added during

-   -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).        Liquefaction Step (a)

According to processes of the invention, liquefaction in step (a) iscarried out by subjecting starch-containing material at a temperatureabove the initial gelatinization temperature, in particular at atemperature between 80-90° C., to an alpha-amylase and optionally aprotease and other enzymes, such as a glucoamylase, a pullulanase and/ora phytase.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch-containing materialcommences. In general, starch heated in water begins to gelatinizebetween about 50° C. and 75° C.; the exact temperature of gelatinizationdepends on the specific starch and can readily be determined by theskilled artisan. Thus, the initial gelatinization temperature may varyaccording to the plant species, to the particular variety of the plantspecies as well as with the growth conditions. In the context of thisinvention the initial gelatinization temperature of a givenstarch-containing material may be determined as the temperature at whichbirefringence is lost in 5% of the starch granules using the methoddescribed by Gorinstein and Lii, 1992, Starch/Stärke 44(12): 461-466.

According to the invention liquefaction in step (a) is typically carriedout at a temperature in the range from 70-100° C. In an embodiment thetemperature in liquefaction is between 75-95° C., such as between 75-90°C., preferably between 80-90° C., such as 82-88° C., such as around 85°C. The pH in liquefaction may be in the range between 3 and 7,preferably from 4 to 6, or more preferably from 4.5 to 5.5.

According to the invention a jet-cooking step may be carried out priorto liquefaction in step (a). The jet-cooking may be carried out at atemperature between 110-145° C., preferably 120-140° C., such as125-135° C., preferably around 130° C. for about 1-15 minutes,preferably for about 3-10 minutes, especially around about 5 minutes.

In an embodiment, the process of the invention further comprises, priorto the liquefaction step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by dry milling;

z) forming a slurry comprising the starch-containing material and water.

According to the invention the dry solid content (DS) in liquefactionlies in the range from 20-55 wt.-%, preferably 25-45 wt.-%, morepreferably 30-40 wt.-% or 30-45 wt-%.

The starch-containing starting material, such as whole grains, may bereduced in particle size, e.g., by milling, in order to open up thestructure, to increase surface area, and allowing for furtherprocessing. Generally there are two types of processes: wet and drymilling. In dry milling whole kernels are milled and used. Wet millinggives a good separation of germ and meal (starch granules and protein).Wet milling is often applied at locations where the starch hydrolysateis used in production of, e.g., syrups. Both dry milling and wet millingare well known in the art of starch processing. According to the presentinvention dry milling is preferred.

In an embodiment the particle size is reduced to between 0.05 to 3.0 mm,preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%,more preferably at least 70%, even more preferably at least 90% of thestarch-containing material fit through a sieve with a 0.05 to 3.0 mmscreen, preferably 0.1-0.5 mm screen. In another embodiment at least50%, preferably at least 70%, more preferably at least 80%, especiallyat least 90% of the starch-containing material fit through a sieve with#6 screen.

Liquefaction in step (a) may be carried out for 0.5-5 hours, such as 1-3hours, such as typically around 2 hours.

The alpha-amylase and other optional enzymes, such as protease, mayinitially be added to the aqueous slurry to initiate liquefaction(thinning). In an embodiment only a portion of the enzymes (e.g., about⅓) is added to the aqueous slurry, while the rest of the enzymes (e.g.,about ⅔) are added in liquefaction step (a).

A non-exhaustive list of examples of alpha-amylases can be found belowin the “Alpha-Amylase Present and/or Added In Liquefaction”-section. Ina preferred embodiment the alpha-amylase is a bacterial alpha-amylase.Bacterial alpha-amylases are typically thermostable. In a preferredembodiment the alpha-amylase is derived from the genus Bacillus, such asa strain of Bacillus stearothermophilus, in particular a variant of aBacillus stearothermophilus alpha-amylase, such as the one shown in SEQID NO: 3 in WO 99/019467 or SEQ ID NO: 8 herein.

In an embodiment the alpha-amylase used in liquefaction step (a) is avariant of the Bacillus stearothermophilus alpha-amylase shown in SEQ IDNO: 8 herein, in particular with the double deletions in I181*+G182*,and optionally with a N193F substitution, and truncated to be around 491amino acids long, e.g., from 480-495 amino acids long.

Examples of suitable Bacillus stearothermophilus alpha-amylase variantscan be found below in the “Thermostable Alpha-Amylase”-section andinclude one from the following group of Bacillus stearothermophilusalpha-amylase variants with double deletions I181*+G182*, and optionallysubstitution N193F, and additionally the following substitutions:

E129V+K177L+R179E;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;

V59A+E129V+K177L+R179E+Q254S+M284V; and

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 8 fornumbering).

According to processes of the invention, liquefaction in step (a) may becarried out using a combination of alpha-amylase (e.g., Bacillusstearothermophilus alpha-amylase shown in SEQ ID NO: 8) and protease(e.g., Pyrococcus furiosus (pfu protease) shown in SEQ ID NO: 9). Aglucoamylase may also be present, such as the one derived fromPenicillium oxalicum shown in SEQ ID NO: 10 herein (see the“Glucoamylase Present and/or Added In Liquefaction Step (a)”—sectionbelow.

Saccharification and Fermentation

A trehalase of the invention, a glucoamylase and optionally a proteaseand/or a cellulolytic enzyme composition may be present and/or added insaccharification step (b); fermentation step (c); simultaneoussaccharification and fermentation (SSF); optionally apresaccharification step before step (b).

In a preferred embodiment the glucoamylase is added together with afungal alpha-amylase, in particular acid fungal alpha-amylase. Examplesof glucoamylases can be found in the “Glucoamylases Present and/or AddedIn Saccharification and/or Fermentation”-section below. When doingsequential saccharification and fermentation, saccharification step (b)may be carried out at conditions well-known in the art, i.e., suitablefor enzyme saccharification. For instance, the saccharification step (b)may last up to from about 24 to about 72 hours.

In an embodiment pre-saccharification is done before saccharification instep (b). Pre-saccharification is typically done for 40-90 minutes at atemperature between 30-65° C., typically about 60° C.Pre-saccharification is in an embodiment followed by saccharificationduring fermentation in simultaneous saccharification and fermentation(SSF). Saccharification is typically carried out at temperatures from20-75° C., preferably from 40-70° C., typically around 60° C., and at apH between 4 and 5, normally at about pH 4.5.

Simultaneous saccharification and fermentation (“SSF”) is widely used inindustrial scale fermentation product production processes, especiallyethanol production processes. When doing SSF the saccharification step(b) and the fermentation step (c) are carried out simultaneously. Thereis no holding stage for the saccharification, meaning that a fermentingorganism, in particular yeast, and enzymes, may be added together.However, it is also contemplated to add the fermenting organism andenzymes separately. SSF is according to the invention typically carriedout at a temperature from 25° C. to 40° C., such as from 28° C. to 35°C., such as from 30° C. to 34° C., preferably around about 32° C. In anembodiment fermentation is ongoing for 6 to 120 hours, in particular 24to 96 hours. In an embodiment the pH is between 4-5.

In an embodiment of the invention a cellulolytic composition is presentand/or added in saccharification step (b), fermentation step (c) orsimultaneous saccharification and fermentation (SSF) orpre-saccharification before step (b). Examples of such cellulolyticcompositions can be found in the “Cellulolytic Enzyme Compositionpresent and/or added during Saccharification and/orFermentation”-section below. The optional cellulolytic enzymecomposition may be present and/or added together with the glucoamylaseand trehalase of the invention. Examples of proteases can be found inthe “Proteases Present and/or Added In Saccharification and/orFermentation”-section below.

In a preferred embodiment the trehalase is present and/or added in anamount between 0.01-20 ug EP trehalase/g DS, such as between 0.05-15 ugEP terhalase/g DS, such as between 0.5 and 10 ug EP trehalase/g DS.

Starch-Containing Materials

According to the invention any suitable starch-containing startingmaterial may be used. The starting material is generally selected basedon the desired fermentation product, in particular ethanol. Examples ofstarch-containing starting materials, suitable for use in processes ofthe present invention, include cereal, tubers or grains. Specificallythe starch-containing material may be corn, wheat, barley, rye, milo,sago, cassava, tapioca, sorghum, oat, rice, peas, beans, or sweetpotatoes, or mixtures thereof. Contemplated are also waxy and non-waxytypes of corn and barley.

In a preferred embodiment the starch-containing starting material iscorn.

In a preferred embodiment the starch-containing starting material iswheat.

In a preferred embodiment the starch-containing starting material isbarley.

In a preferred embodiment the starch-containing starting material isrye.

In a preferred embodiment the starch-containing starting material ismilo.

In a preferred embodiment the starch-containing starting material issago.

In a preferred embodiment the starch-containing starting material iscassava.

In a preferred embodiment the starch-containing starting material istapioca.

In a preferred embodiment the starch-containing starting material issorghum.

In a preferred embodiment the starch-containing starting material isrice,

In a preferred embodiment the starch-containing starting material ispeas.

In a preferred embodiment the starch-containing starting material isbeans.

In a preferred embodiment the starch-containing starting material issweet potatoes.

In a preferred embodiment the starch-containing starting material isoats.

Producing a Fermentation Product from Ungelatinized Starch MaterialUsing a Trehalase of the Invention

A trehalase of the invention may suitably be used in a raw starchhydrolysis (RSH) process for producing desired fermentation products, inparticular ethanol. In RSH processes the starch does not gelatinize asthe process is carried out at temperatures below the initialgelatinization temperature of the starch in question (defined above).

The desired fermentation product may in an embodiment be ethanolproduced from ungelatinized (i.e., uncooked), preferably milled, grains,such as corn, or small grains such as wheat, oats, barley, rye, rice, orcereals such as sorghum. Examples of suitable starch-containing startingmaterials are listed in the section “Starch-ContainingMaterials”-section above.

Accordingly, in this aspect the invention relates to processes ofproducing fermentation products from starch-containing materialcomprising:

(a) saccharifying a starch-containing material at a temperature belowthe initial gelatinization temperature; and

(b) fermenting using a fermentation organism; and

(c) optionally recovering the fermentation product;

wherein saccharification and/or fermentation is done in the presence ofthe following enzymes: glucoamylase, alpha-amylase, trehalase of theinvention, and optionally a cellulolytic enzyme composition and/or aprotease.

Before step (a) an aqueous slurry of starch-containing material, such asgranular starch, having 10-55 wt.-% dry solids (DS), preferably 25-45wt.-% dry solids, more preferably 30-40% dry solids of starch-containingmaterial may be prepared. The slurry may include water and/or processwaters, such as stillage (backset), scrubber water, evaporatorcondensate or distillate, side-stripper water from distillation, orprocess water from other fermentation product plants. Because a rawstarch hydrolysis process of the invention is carried out below theinitial gelatinization temperature, and thus no significant viscosityincrease takes place, high levels of stillage may be used, if desired.In an embodiment the aqueous slurry contains from about 1 to about 70vol.-%, preferably 15-60% vol.-%, especially from about 30 to 50 vol.-%water and/or process waters, such as stillage (backset), scrubber water,evaporator condensate or distillate, side-stripper water fromdistillation, or process water from other fermentation product plants,or combinations thereof, or the like.

In an embodiment backset, or another recycled stream, is added to theslurry before step (a), or to the saccharification (step (a)), or to thesimultaneous saccharification and fermentation steps (combined step (a)and step (b)).

A RSH process of the invention is conducted at a temperature below theinitial gelatinization temperature, which means that the temperature atwhich a separate step (a) is carried out typically lies in the rangebetween 25-75° C., such as between 30-70° C., or between 45-60° C.

In a preferred embodiment the temperature during fermentation in step(b) or simultaneous saccharification and fermentation in steps (a) and(b) is between 25° C. and 40° C., preferably between 28° C. and 36° C.,such as between 28° C. and 35° C., such as between 28° C. and 34° C.,such as around 32° C.

In an embodiment of the invention fermentation is carried out for 30 to150 hours, preferably 48 to 96 hours. 66.

In an embodiment fermentation is carried out so that the sugar level,such as glucose level, is kept at a low level, such as below 6 wt.-%,such as below about 3 wt.-%, such as below about 2 wt.-%, such as belowabout 1 wt.-%., such as below about 0.5%, or below 0.25% wt.-%, such asbelow about 0.1 wt.-%. Such low levels of sugar can be accomplished bysimply employing adjusted quantities of enzymes and fermenting organism.A skilled person in the art can easily determine which doses/quantitiesof enzyme and fermenting organism to use. The employed quantities ofenzyme and fermenting organism may also be selected to maintain lowconcentrations of maltose in the fermentation broth. For instance, themaltose level may be kept below about 0.5 wt.-%, such as below about 0.2wt.-%.

The process of the invention may be carried out at a pH from 3 and 7,preferably from 3 to 6, or more preferably from 3.5 to 5.0.

The term “granular starch” means raw uncooked starch, i.e., starch inits natural form found in, e.g., cereal, tubers or grains. Starch isformed within plant cells as tiny granules insoluble in water. When putin cold water, the starch granules may absorb a small amount of theliquid and swell. At temperatures up to around 50° C. to 75° C. theswelling may be reversible. However, at higher temperatures anirreversible swelling called “gelatinization” begins. The granularstarch may be a highly refined starch, preferably at least 90%, at least95%, at least 97% or at least 99.5% pure, or it may be a more crudestarch-containing materials comprising (e.g., milled) whole grainsincluding non-starch fractions such as germ residues and fibers.

The raw material, such as whole grains, may be reduced in particle size,e.g., by milling, in order to open up the structure and allowing forfurther processing. Examples of suitable particle sizes are disclosed inU.S. Pat. No. 4,514,496 and WO2004/081193 (both references areincorporated by reference). Two processes are preferred according to theinvention: wet and dry milling. In dry milling whole kernels are milledand used. Wet milling gives a good separation of germ and meal (starchgranules and protein) and is often applied at locations where the starchhydrolysate is used in production of, e.g., syrups. Both dry and wetmilling is well known in the art of starch processing.

In an embodiment the particle size is reduced to between 0.05 to 3.0 mm,preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%,more preferably at least 70%, even more preferably at least 90% of thestarch-containing material fit through a sieve with a 0.05 to 3.0 mmscreen, preferably 0.1-0.5 mm screen. In a preferred embodimentstarch-containing material is prepared by reducing the particle size ofthe starch-containing material, preferably by milling, such that atleast 50% of the starch-containing material has a particle size of0.1-0.5 mm.

In a preferred embodiment the trehalase is present and/or added in anamount between 0.01-20 ug EP trehalase/g DS, such as between 0.05-15 ugEP terhalase/g DS, such as between 0.5 and 10 ug EP trehalase/g DS.

According to the invention the enzymes are added so that theglucoamylase is present in an amount of 0.001 to 10 AGU/g DS, preferablyfrom 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS.

According to the invention the enzymes are added so that thealpha-amylase is present or added in an amount of 0.001 to 10 AFAU/g DS,preferably from 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

According to the invention the enzymes are added so that thecellulolytic enzyme composition is present or added in an amount1-10,000 micro grams EP/g DS, such as 2-5,000, such as 3 and 1,000, suchas 4 and 500 micro grams EP/g DS.

According to the invention the enzymes are added so that thecellulolytic enzyme composition is present or added in an amount in therange from 0.1-100 FPU per gram total solids (TS), preferably 0.5-50 FPUper gram TS, especially 1-20 FPU per gram TS.

In an embodiment of the invention the enzymes are added so that theprotease is present in an amount of 0.0001-1 mg enzyme protein per g DS,preferably 0.001 to 0.1 mg enzyme protein per g DS. Alternatively, theprotease is present and/or added in an amount of 0.0001 to 1 LAPU/g DS,preferably 0.001 to 0.1 LAPU/g DS and/or 0.0001 to 1 mAU-RH/g DS,preferably 0.001 to 0.1 mAU-RH/g DS.

In an embodiment of the invention the enzymes are added so that theprotease is present or added in an amount in the range 1-1,000 μg EP/gDS, such as 2-500 μg EP/g DS, such as 3-250 μg EP/g DS.

In a preferred embodiment ratio between glucoamylase and alpha-amylaseis between 99:1 and 1:2, such as between 98:2 and 1:1, such as between97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5,94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg EP glucoamylase: mg EPalpha-amylase).

In a preferred embodiment the total dose of glucoamylase andalpha-amylase is according to the invention from 10-1,000 μg/g DS, suchas from 50-500 μg/g DS, such as 75-250 μg/g DS.

In a preferred embodiment the total dose of cellulolytic enzymecomposition added is from 10-500 μg/g DS, such as from 20-400 μg/g DS,such as 20-300 μg/g DS.

In an embodiment the glucoamylase, such as one derived from Trametescingulata, used in fermentation or SSF exhibits at least 60%, such as atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% oreven 100% identity to the mature part of SEQ ID NO: 11.

In an embodiment the glucoamylase, such as one derived from Pycnoporussanguineus, used in fermentation or SSF exhibits at least 60%, such asat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% oreven 100% identity to the mature part of SEQ ID NO: 12.

In an embodiment the alpha-amylase used in fermentation or SSF exhibitsat least 60%, such as at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or even 100% identity to the mature part of SEQ ID NO:7.

In a preferred embodiment the invention relates to processes ofproducing fermentation products from starch-containing materialcomprising:

(a) saccharifying a starch-containing material at a temperature belowthe initial gelatinization temperature; and

(b) fermenting using a fermentation organism;

wherein saccharification and/or fermentation is done in the presence ofthe following enzymes:

i) glucoamylase;

ii) alpha-amylase;

iii) trehalse of the invention;

iii) optionally a cellulolytic enzyme composition and/or a protease.

In a preferred embodiment the enzymes may be added as an enzymecomposition of the invention. In a preferred embodiment steps (a) and(b) are carried out simultaneously (i.e., one-step fermentation).However, step (a) and (b) may also be carried our sequentially.

Fermentation

Fermentation is carried out in a fermentation medium. The fermentationmedium includes the fermentation substrate, that is, the carbohydratesource that is metabolized by the fermenting organism. According to theinvention the fermentation medium may comprise nutrients and growthstimulator(s) for the fermenting organism(s). Nutrient and growthstimulators are widely used in the art of fermentation and includenitrogen sources, such as ammonia; urea, vitamins and minerals, orcombinations thereof.

Fermenting Organisms for Starch Based Fermentation

The term “fermenting organism” refers to any organism, includingbacterial and fungal organisms, especially yeast, suitable for use in afermentation process and capable of producing the desired fermentationproduct. Especially suitable fermenting organisms are able to ferment,i.e., convert, sugars, such as glucose or maltose, directly orindirectly into the desired fermentation product, such as in particularethanol. Examples of fermenting organisms include fungal organisms, suchas in particular yeast. Preferred yeast includes strains ofSaccharomyces spp., in particular, Saccharomyces cerevisiae. In aparticular embodiment the S. cerevisiae expresses the trehalase of theinvention.

Suitable concentrations of the viable fermenting organism duringfermentation, such as SSF, are well known in the art or can easily bedetermined by the skilled person in the art. In one embodiment thefermenting organism, such as ethanol fermenting yeast, (e.g.,Saccharomyces cerevisiae) is added to the fermentation medium so thatthe viable fermenting organism, such as yeast, count per mL offermentation medium is in the range from 10⁵ to 10¹², preferably from10⁷ to 10¹⁰, especially about 5×10⁷.

Examples of commercially available yeast includes, e.g., RED STAR™ andETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI(available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

Recovery

Subsequent to fermentation, e.g., SSF, the fermentation product, inparticular ethanol may be separated from the fermentation medium. Theslurry may be distilled to recover/extract the desired fermentationproduct (i.e., ethanol). Alternatively the desired fermentation product(i.e., ethanol) may be extracted from the fermentation medium by microor membrane filtration techniques. The fermentation product (i.e.,ethanol) may also be recovered by stripping or other method well knownin the art.

Alpha-Amylase Present and/or Added in Liquefaction

According to the invention an alpha-amylase is present and/or added inliquefaction optionally together with other enzymes such as a protease,a glucoamylase, phytase and/or pullulanase. The alpha-amylase added inliquefaction step (a) may be any alpha-amylase. Preferred are bacterialalpha-amylases, which typically are stable at temperatures used inliquefaction.

Bacterial Alpha-Amylase

The term “bacterial alpha-amylase” means any bacterial alpha-amylaseclassified under EC 3.2.1.1. A bacterial alpha-amylase used according tothe invention may, e.g., be derived from a strain of the genus Bacillus,which is sometimes also referred to as the genus Geobacillus. In anembodiment the Bacillus alpha-amylase is derived from a strain ofBacillus amyloliquefaciens, Bacillus licheniformis, Bacillusstearothermophilus, or Bacillus subtilis, but may also be derived fromother Bacillus sp.

Specific examples of bacterial alpha-amylases include the Bacillusstearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQID NO: 8 herein, the Bacillus amyloliquefaciens alpha-amylase of SEQ IDNO: 5 in WO 99/19467, and the Bacillus licheniformis alpha-amylase ofSEQ ID NO: 4 in WO 99/19467 (all sequences are hereby incorporated byreference). In an embodiment the alpha-amylase may be an enzyme having adegree of identity of at least 60%, e.g., at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% to any of the sequences shown in SEQ ID NOS: 3, 4 or 5,respectively, in WO 99/19467.

In an embodiment the alpha-amylase may be an enzyme having a degree ofidentity of at least 60%, e.g., at least 70%, at least 80%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% to any ofthe sequences shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 8herein.

In a preferred embodiment the alpha-amylase is derived from Bacillusstearothermophilus. The Bacillus stearothermophilus alpha-amylase may bea mature wild-type or a mature variant thereof. The mature Bacillusstearothermophilus alpha-amylases may naturally be truncated duringrecombinant production. For instance, the Bacillus stearothermophilusalpha-amylase may be a truncated so it has around 491 amino acids, e.g.,so that it is between 480-495 amino acids long, so it lacks a functionalstarch binding domain (compared to SEQ ID NO: 3 in WO 99/19467) or SEQID NO: 8 herein.

The Bacillus alpha-amylase may also be a variant and/or hybrid. Examplesof such a variant can be found in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents arehereby incorporated by reference). Specific alpha-amylase variants aredisclosed in U.S. Pat. Nos. 6,093,562, 6,187,576, 6,297,038, and7,713,723 (hereby incorporated by reference) and include Bacillusstearothermophilus alpha-amylase (often referred to as BSGalpha-amylase) variants having a deletion of one or two amino acids atpositions R179, G180, I181 and/or G182, preferably a double deletiondisclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (herebyincorporated by reference), preferably corresponding to deletion ofpositions I181 and G182 compared to the amino acid sequence of Bacillusstearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed inWO 99/19467 or SEQ ID NO: 8 herein or the deletion of amino acids R179and G180 using SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 8 herein fornumbering (which reference is hereby incorporated by reference). Evenmore preferred are Bacillus alpha-amylases, especially Bacillusstearothermophilus alpha-amylases, which have a double deletioncorresponding to a deletion of positions 181 and 182 and furthercomprise a N193F substitution (also denoted I181*+G182*+N193F) comparedto the wild-type BSG alpha-amylase amino acid sequence set forth in SEQID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 8 herein. The bacterialalpha-amylase may also have a substitution in a position correspondingto S239 in the Bacillus licheniformis alpha-amylase shown in SEQ ID NO:4 in WO 99/19467, or a S242 and/or E188P variant of the Bacillusstearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQID NO: 8 herein.

In an embodiment the variant is a S242A, E or Q variant, preferably aS242Q variant, of the Bacillus stearothermophilus alpha-amylase (usingSEQ ID NO: 8 herein for numbering).

In an embodiment the variant is a position E188 variant, preferablyE188P variant of the Bacillus stearothermophilus alpha-amylase (usingSEQ ID NO: 8 herein for numbering).

The bacterial alpha-amylase may in an embodiment be a truncated Bacillusalpha-amylase. Especially the truncation is so that, e.g., the Bacillusstearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 orSEQ ID NO: 8 herein, is around 491 amino acids long, such as from 480 to495 amino acids long, or so it lack a functional starch binding domain.

Bacterial Hybrid Alpha-Amylases

The bacterial alpha-amylase may also be a hybrid bacterialalpha-amylase, e.g., an alpha-amylase comprising 445 C-terminal aminoacid residues of the Bacillus licheniformis alpha-amylase (shown in SEQID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues ofthe alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQID NO: 5 of WO 99/19467). In a preferred embodiment this hybrid has oneor more, especially all, of the following substitutions:

G48A+T491+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacilluslicheniformis numbering in SEQ ID NO: 4 of WO 99/19467). Also preferredare variants having one or more of the following mutations (orcorresponding mutations in other Bacillus alpha-amylases): H154Y, A181T,N190F, A209V and Q264S and/or the deletion of two residues betweenpositions 176 and 179, preferably the deletion of E178 and G179 (usingSEQ ID NO: 5 of WO 99/19467 for position numbering).

In an embodiment the bacterial alpha-amylase is the mature part of thechimeric alpha-amylase disclosed in Richardson et al. (2002), TheJournal of Biological Chemistry, Vol. 277, No 29, Issue 19 July, pp.267501-26507, referred to as BD5088 or a variant thereof. Thisalpha-amylase is the same as the one shown in SEQ ID NO: 2 in WO2007134207. The mature enzyme sequence starts after the initial “Met”amino acid in position 1.

Thermostable Alpha-Amylase

The alpha-amylase may be a thermostable alpha-amylase, such as athermostable bacterial alpha-amylase, preferably from Bacillusstearothermophilus.

In an embodiment of the invention the alpha-amylase is an bacterialalpha-amylase, preferably derived from the genus Bacillus, especially astrain of Bacillus stearothermophilus, in particular the Bacillusstearothermophilus as disclosed in WO 99/019467 as SEQ ID NO: 3 (SEQ IDNO: 8 herein) with one or two amino acids deleted at positions R179,G180, I181 and/or G182, in particular with R179 and G180 deleted, orwith I181 and G182 deleted, with mutations in below list of mutations.

In preferred embodiments the Bacillus stearothermophilus alpha-amylaseshave double deletion I181+G182, and optionally substitution N193F,further comprising mutations selected from below list:

V59A + Q89R + G112D + E129V + K177L + R179E + K220P + N224L + Q254S;V59A + Q89R + E129V + K177L + R179E + H208Y + K220P + N224L + Q254S;V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E +D281N; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +I270L; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +H274K; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +Y276F; V59A + E129V + R157Y + K177L + R179E + K220P + N224L + S242Q +Q254S; V59A + E129V + K177L + R179E + H208Y + K220P + N224L + S242Q +Q254S; 59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274K;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + Y276F;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + D281N;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + M284T;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + G416V;V59A + E129V + K177L + R179E + K220P + N224L + Q254S; V59A + E129V +K177L + R179E + K220P + N224L + Q254S + M284T; A91L + M96I + E129V +K177L + R179E + K220P + N224L + S242Q + Q254S; E129V + K177L + R179E;E129V + K177L + R179E + K220P + N224L + S242Q + Q254S; E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + Y276F + L427M; E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + M284T; E129V + K177L + R179E +K220P + N224L + S242Q +Q254S + N376* + I377*; E129V + K177L + R179E +K220P + N224L + Q254S; E129V + K177L + R179E + K220P + N224L + Q254S +M284T; E129V + K177L + R179E + S242Q; E129V + K177L + R179V + K220P +N224L + S242Q + Q254S; K220P + N224L + S242Q + Q254S; M284V; V59A +Q89R + E129V + K177L + R179E + Q254S + M284V. V59A + E129V + K177L +R179E + Q254S + M284V;

Specific information about the thermostability of above alpha-amylasesvariants can be found in WO12/088303 (Novozymes) which is herebyincorporated by reference.

In a preferred embodiment the alpha-amylase is selected from the groupof Bacillus stearothermophilus alpha-amylase variants having a doubledeletion in I181+G182, and optionally a substitution in N193F, andsubstitutions from the following list

E129V+K177L+R179E;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;

V59A+E129V+K177L+R179E+Q254S+M284V; and

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 8 herein fornumbering).

It should be understood that when referring to Bacillusstearothermophilus alpha-amylase and variants thereof they are normallyproduced in truncated form. In particular, the truncation may be so thatthe Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 inWO 99/19467 or SEQ ID NO: 8 herein, or variants thereof, are truncatedin the C-terminal and are typically around 491 amino acids long, such asfrom 480-495 amino acids long, or so that it lacks a functional starchbinding domain.

In a preferred embodiment the alpha-amylase variant may be an enzymehaving a degree of identity of at least 60%, e.g., at least 70%, atleast 80%, at least 90%, at least 95%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99%, but less than 100% to the sequence shown inSEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 8 herein.

In an embodiment the bacterial alpha-amylase, e.g., Bacillusalpha-amylase, such as especially Bacillus stearothermophilusalpha-amylase, or variant thereof, is dosed to liquefaction in aconcentration between 0.01-10 KNU-A/g DS, e.g., between 0.02 and 5KNU-A/g DS, such as 0.03 and 3 KNU-A, preferably 0.04 and 2 KNU-A/g DS,such as especially 0.01 and 2 KNU-A/g DS. In an embodiment the bacterialalpha-amylase, e.g., Bacillus alpha-amylase, such as especially Bacillusstearothermophilus alpha-amylases, or variant thereof, is dosed toliquefaction in a concentration of between 0.0001-1 mg EP (EnzymeProtein)/g DS, e.g., 0.0005-0.5 mg EP/g DS, such as 0.001-0.1 mg EP/gDS.

Protease Present and/or Added in Liquefaction

According to the invention a protease may optionally be present and/oradded in liquefaction together with the alpha-amylase, and an optionalglucoamylase, phytase and/or pullulanase.

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

In a preferred embodiment the thermostable protease used according tothe invention is a “metallo protease” defined as a protease belonging toEC 3.4.24 (metalloendopeptidases); preferably EC 3.4.24.39 (acid metalloproteinases).

To determine whether a given protease is a metallo protease or not,reference is made to the above “Handbook of Proteolytic Enzymes” and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any suitable assay, in which asubstrate is employed, that includes peptide bonds relevant for thespecificity of the protease in question. Assay-pH and assay-temperatureare likewise to be adapted to the protease in question. Examples ofassay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples ofassay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80° C.

Examples of protease substrates are casein, such as Azurine-CrosslinkedCasein (AZCL-casein). Two protease assays are described below in the“Materials & Methods”-section, of which the so-called “AZCL-CaseinAssay” is the preferred assay.

There are no limitations on the origin of the protease used in a processof the invention as long as it fulfills the thermostability propertiesdefined below.

In one embodiment the protease is of fungal origin.

The protease may be a variant of, e.g., a wild-type protease as long asthe protease is thermostable. In a preferred embodiment the thermostableprotease is a variant of a metallo protease as defined above. In anembodiment the thermostable protease used in a process of the inventionis of fungal origin, such as a fungal metallo protease, such as a fungalmetallo protease derived from a strain of the genus Thermoascus,preferably a strain of Thermoascus aurantiacus, especially Thermoascusaurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39).

In an embodiment the thermostable protease is a variant of the maturepart of the metallo protease shown in SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 andshown as SEQ ID NO: 13 herein, further with mutations selected frombelow list:

S5*+D79L+S87P+A112P+D142L;

D79L+S87P+A 112P+T124V+D142L;

S5*+N26R+D79L+S87P+A112P+D142L;

N26R+T46R+D79L+S87P+A112P+D142L;

T46R+D79L+S87P+T116V+D142L;

D79L+P81R+S87P+A112P+D142L;

A27K+D79L+S87P+A112P+T124V+D142L;

D79L+Y82F+S87P+A112P+T124V+D142L;

D79L+Y82F+S87P+A112P+T124V+D142L;

D79L+S87P+A112P+T124V+A126V+D142L;

D79L+S87P+A112P+D142L;

D79L+Y82F+S87P+A112P+D142L;

S38T+D79L+S87P+A112P+A126V+D142L;

D79L+Y82F+S87P+A112P+A126V+D142L;

A27K+D79L+S87P+A112P+A126V+D142L;

D79L+S87P+N98C+A112P+G135C+D142L;

D79L+S87P+A112P+D142L+T141C+M161C;

S36P+D79L+S87P+A112P+D142L;

A37P+D79L+S87P+A112P+D142L;

S49P+D79L+S87P+A112P+D142L;

S50P+D79L+S87P+A112P+D142L;

D79L+S87P+D104P+A112P+D142L;

D79L+Y82F+S87G+A112P+D142L;

S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;

D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;

S70V+D79L+Y82F+S87G+A112P+D142L;

D79L+Y82F+S87G+D104P+A112P+D142L;

D79L+Y82F+S87G+A112P+A126V+D142L;

Y82F+S87G+S70V+D79L+D104P+A112P+D142L;

Y82F+S87G+D79L+D104P+A112P+A126V+D142L;

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;

A27K+Y82F+S87G+D104P+A112P+A126V+D142L;

A27K+D79L+Y82F+D104P+A112P+A126V+D142L;

A27K+Y82F+D104P+A112P+A126V+D142L;

A27K+D79L+S87P+A112P+D142L;

D79L+S87P+D142L.

Specific information about the thermostability of above proteasevariants can be found in WO12/088303 (Novozymes), which is herebyincorporated by reference.

In an preferred embodiment the thermostable protease is a variant of themetallo protease disclosed as the mature part of SEQ ID NO: 2 disclosedin WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841or SEQ ID NO: 13 herein with the following mutations:

D79L+S87P+A112P+D142L;

D79L+S87P+D142L; or

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

In an embodiment the protease variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQID NO: 13 herein.

The thermostable protease may also be derived from any bacterium as longas the protease has the thermostability properties defined according tothe invention.

In an embodiment the thermostable protease is derived from a strain ofthe bacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfuprotease).

In an embodiment the protease is one shown as SEQ ID NO: 1 in U.S. Pat.No. 6,358,726-B1 (Takara Shuzo Company), or SEQ ID NO: 9 herein.

In another embodiment the thermostable protease is one disclosed in SEQID NO: 22 herein or a protease having at least 80% identity, such as atleast 85%, such as at least 90%, such as at least 95%, such as at least96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-B1 or SEQ ID NO: 9herein. Pyroccus furiosus protease can be purchased from Takara Bio,Japan.

Glucoamylase Present and/or Added in Liquefaction

According to the invention a glucoamylase may optionally be presentand/or added in liquefaction step (a). In a preferred embodiment theglucoamylase is added together with or separately from the alpha-amylaseand optional protease, phytase and/or pullulanase.

In a specific and preferred embodiment the glucoamylase, preferably offungal origin, preferably a filamentous fungi, is from a strain of thegenus Penicillium, especially a strain of Penicillium oxalicum, inparticular the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in WO 2011/127802 (which is hereby incorporated by reference) andshown in SEQ ID NO: 10 herein.

In an embodiment the glucoamylase has at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 91%,more preferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99% or 100%identity to the mature polypeptide shown in SEQ ID NO: 2 in WO2011/127802 or SEQ ID NO: 10 herein.

In a preferred embodiment the glucoamylase is a variant of thePenicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO2011/127802 and shown in SEQ ID NO: 10 herein, having a K79Vsubstitution (using the mature sequence shown in SEQ ID NO: 10 hereinfor numbering). The K79V glucoamylase variant has reduced sensitivity toprotease degradation relative to the parent as disclosed in WO2013/036526 (which is hereby incorporated by reference).

In an embodiment the glucoamylase is derived from Penicillium oxalicum.

In an embodiment the glucoamylase is a variant of the Penicilliumoxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 andshown in SEQ ID NO: 10 herein. In a preferred embodiment the Penicilliumoxalicum glucoamylase is the one disclosed as SEQ ID NO: 2 in WO2011/127802 and shown in SEQ ID NO: 10 herein having Val (V) in position79 (using SEQ ID NO: 23 herein for numbering).

Contemplated Penicillium oxalicum glucoamylase variants are disclosed inWO 2013/053801 which is hereby incorporated by reference.

In an embodiment these variants have reduced sensitivity to proteasedegradation.

In an embodiment these variant have improved thermostability compared tothe parent.

More specifically, in an embodiment the glucoamylase has a K79Vsubstitution (using SEQ ID NO: 10 herein for numbering), (PE001variant), and further comprises at least one of the followingsubstitutions or combination of substitutions:

T65A; or

Q327F; or

E501V; or

Y504T; or

Y504*; or

T65A+Q327F; or

T65A+E501V; or

T65A+Y504T; or

T65A+Y504*; or

Q327F+E501V; or

Q327F+Y504T; or

Q327F+Y504*; or

E501V+Y504T; or

E501V+Y504*; or

T65A+Q327F+E501V; or

T65A+Q327F+Y504T; or

T65A+E501V+Y504T; or

Q327F+E501V+Y504T; or

T65A+Q327F+Y504*; or

T65A+E501V+Y504*; or

Q327F+E501V+Y504*; or

T65A+Q327F+E501V+Y504T; or

T65A+Q327F+E501V+Y504*;

E501V+Y504T; or

T65A+K161S; or

T65A+Q405T; or

T65A+Q327W; or

T65A+Q327F; or

T65A+Q327Y; or

P11F+T65A+Q327F; or

R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F; or

P11F+D26C+K33C+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or

R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or

P11F+T65A+Q327W; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P11F+T65A+Q327W+E501V+Y504T; or

T65A+Q327F+E501V+Y504T; or

T65A+S105P+Q327W; or

T65A+S105P+Q327F; or

T65A+Q327W+S364P; or

T65A+Q327F+S364P; or

T65A+S103N+Q327F; or

P2N+P4S+P11F+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S; or

P2N+P4S+P11F+T65A+1172V+Q327F; or

P2N+P4S+P11F+T65A+Q327F+N502*; or

P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; or

P2N+P4S+P11F+T65A+Q327F+S377T; or

P2N+P4S+P11F+T65A+V325T+Q327W; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+T65A+1172V+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+1375A+E501V+Y504T; or

P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; or

P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; or

P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+V791+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or

S255N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

In a preferred embodiment the Penicillium oxalicum glucoamylase varianthas a K79V substitution (using SEQ ID NO: 10 herein for numbering),corresponding to the PE001 variant, and further comprises one of thefollowing mutations:

P11F+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F; or

P11F+D26C+K33C+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P11F+T65A+Q327W+E501V+Y504T.

The glucoamylase may be added in amounts from 0.1-100 micrograms EP/g,such as 0.5-50 micrograms EP/g, such as 1-25 micrograms EP/g, such as2-12 micrograms EP/g DS.

Trehalase Present and/or Added in Saccharification and/or Fermentation

According to the process of the invention a trehalase of the inventionis present and/or added during the

-   -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).

In a preferred embodiment the mature trehalase disclosed in SEQ ID NO:21. In a preferred embodiment the mature trehalase disclosed in SEQ IDNO: 23. In a preferred embodiment the trehalase is present and/or addedin an amount between 0.01-20 ug EP (Enzyme Protein) trehalase/g DS, suchas between 0.05-15 ug EP terhalase/g DS, such as between 0.5 and 10 ugEP trehalase/g DS.

Glucoamylase Present and/or Added in Saccharification and/orFermentation

The glucoamylase present and/or added during saccharification step (b);fermentation step (c); simultaneous saccharification and fermentation;or presaccharification before step (b), may be derived from any suitablesource, e.g., derived from a microorganism or a plant. Preferredglucoamylases are of fungal or bacterial origin, selected from the groupconsisting of Aspergillus glucoamylases, in particular Aspergillus nigerG1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102),or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylasedisclosed in WO 84/02921, Aspergillus oryzae glucoamylase (Agric. Biol.Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.Other Aspergillus glucoamylase variants include variants with enhancedthermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9,499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582);N182 (Chen et al. (1994), Biochem. J. 301, 275-281); disulphide bonds,A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; andintroduction of Pro residues in position A435 and S436 (Li et al.(1997), Protein Eng. 10, 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and(Nagasaka et al. (1998) “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, ApplMicrobiol Biotechnol 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (US patent no. Re. 32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No. 4,587,215). In a preferred embodiment theglucoamylase used during saccharification and/or fermentation is theTalaromyces emersonii glucoamylase disclosed in WO 99/28448.

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831). Contemplated fungalglucoamylases include Trametes cingulate (SEQ ID NO: 11), Pachykytosporapapyracea; and Leucopaxillus giganteus all disclosed in WO 2006/069289;or Peniophora rufomarginata disclosed in WO2007/124285; or a mixturethereof. Also hybrid glucoamylase are contemplated according to theinvention. Examples include the hybrid glucoamylases disclosed in WO2005/045018. Specific examples include the hybrid glucoamylase disclosedin Table 1 and 4 of Example 1 (which hybrids are hereby incorporated byreference).

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus sanguineus as describedin WO 2011/066576 (SEQ ID NOs 2, 4 or 6), in particular the one shown aSEQ ID NO: 12 herein (corresponding to SEQ ID NO: 4 in WO 2011/066576)or from a strain of the genus Gloeophyllum, such as a strain ofGloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strainof Gloeophyllum as described in WO 2011/068803 (SEQ ID NO: 2, 4, 6, 8,10, 12, 14 or 16). In a preferred embodiment the glucoamylase is SEQ IDNO: 2 in WO 2011/068803 or SEQ ID NO: 5 herein (i.e. Gloeophyllumsepiarium glucoamylase). In a preferred embodiment the glucoamylase isSEQ ID NO: 6 herein (i.e., Gloeophyllum trabeum glucoamylase disclosesas SEQ ID NO: 3 in WO2014/177546) (all references hereby incorporated byreference).

Contemplated are also glucoamylases which exhibit a high identity to anyof the above mentioned glucoamylases, i.e., at least 60%, such as atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% oreven 100% identity to any one of the mature parts of the enzymesequences mentioned above, such as any of SEQ ID NOs: 4, 11, 5, 6 or 12herein, respectively.

In an embodiment the glucoamylase used in fermentation or SSF exhibitsat least 60%, such as at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or even 100% identity to the mature part of SEQ ID NO:6 herein.

In an embodiment the glucoamylase used in fermentation or SSF exhibitsat least 60%, such as at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or even 100% identity to the mature part of SEQ ID NO:12 herein.

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2AGU/g DS.

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 1-1,000 μg EP/g DS, preferably10-500 μg/gDS, especially between 25-250 μg/g DS.

In an embodiment the glucoamylase is added as a blend further comprisingan alpha-amylase.

In a preferred embodiment the alpha-amylase is a fungal alpha-amylase,especially an acid fungal alpha-amylase. The alpha-amylase is typicallya side activity.

In an embodiment the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in WO 99/28448 as SEQ ID NO: 34 or SEQID NO: 4 herein and Trametes cingulata glucoamylase disclosed as SEQ IDNO: 2 in WO 06/069289 and SEQ ID NO: 11 herein.

In an embodiment the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in SEQ ID NO: 4 herein, Trametescingulata glucoamylase disclosed as SEQ ID NO: 11 herein, and Rhizomucorpusillus alpha-amylase with Aspergillus niger glucoamylase linker andSBD disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 7herein.

In an embodiment the glucoamylase is a blend comprising Gloeophyllumsepiarium glucoamylase shown as SEQ ID NO: 5 herein and Rhizomucorpusillus alpha-amylase with an Aspergillus niger glucoamylase linker andstarch-binding domain (SBD), disclosed SEQ ID NO: 7 herein with thefollowing substitutions: G128D+D143N.

In an embodiment the alpha-amylase may be derived from a strain of thegenus Rhizomucor, preferably a strain the Rhizomucor pusillus, such asthe one shown in SEQ ID NO: 3 in WO2013/006756, or the genus Meripilus,preferably a strain of Meripilus giganteus. In a preferred embodimentthe alpha-amylase is derived from a Rhizomucor pusillus with anAspergillus niger glucoamylase linker and starch-binding domain (SBD),disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 7 herein.

In an embodiment the Rhizomucor pusillus alpha-amylase or the Rhizomucorpusillus alpha-amylase with a linker and starch-binding domain (SBD),preferably Aspergillus niger glucoamylase linker and SBD, has at leastone of the following substitutions or combinations of substitutions:D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W;G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W;N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C;Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N;Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R;Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; orG128D+Y141W+D143N+K192R+P219C (using SEQ ID NO: 3 in WO 2013/006756 fornumbering or SEQ ID NO: 7 herein). In a preferred embodiment theglucoamylase blend comprises Gloeophyllum sepiarium glucoamylase (e.g.,SEQ ID NO: 2 in WO 2011/068803 or SEQ ID NO: 5 herein) and Rhizomucorpusillus alpha-amylase.

In a preferred embodiment the glucoamylase blend comprises Gloeophyllumsepiarium glucoamylase shown as SEQ ID NO: 2 in WO 2011/068803 or SEQ IDNO: 5 herein and Rhizomucor pusillus with a linker and starch-bindingdomain (SBD), preferably Aspergillus niger glucoamylase linker and SBD,disclosed SEQ ID NO: 3 in WO 2013/006756 and SEQ ID NO: 7 herein withthe following substitutions: G128D+D143N.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL, SPIRIZYMEACHIEVE™, and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417(from DuPont-Danisco); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™G900, G-ZYME™ and G990 ZR (from DuPont-Danisco).

Cellulolytic Enzyme Composition Present and/or Added in Saccharificationand/or Fermentation

According to the invention a cellulolytic enzyme composition may bepresent in saccharification, fermentation or simultaneoussaccharification and fermentation (SSF).

The cellulolytic enzyme composition comprises a beta-glucosidase, acellobiohydrolase and an endoglucanase.

Examples of suitable cellulolytic composition can be found in WO2008/151079 and WO 2013/028928 which are incorporated by reference.

In preferred embodiments the cellulolytic enzyme composition is derivedfrom a strain of Trichoderma, Humicola, or Chrysosporium.

In an embodiment the cellulolytic enzyme composition is derived from astrain of Trichoderma reesei, Humicola insolens and/or Chrysosporiumlucknowense.

In an embodiment the cellulolytic enzyme composition comprises abeta-glucosidase, preferably one derived from a strain of the genusAspergillus, such as Aspergillus oryzae, such as the one disclosed in WO2002/095014 or the fusion protein having beta-glucosidase activitydisclosed in WO 2008/057637 (in particular the Aspergillus oryzaebeta-glucosidase variant fusion protein shown in SEQ ID NOs: 73 and 74,respectively, in WO 2008/057637 or the Aspergillus oryzaebeta-glucosidase fusion protein shown in SEQ ID NOs: 75 and 76,respectively, in WO 2008/057637—both hereby incorporated by reference),or Aspergillus fumigatus, such as one disclosed in WO 2005/047499 or SEQID NO: 14 herein or an Aspergillus fumigatus beta-glucosidase variantdisclosed in WO 2012/044915 (Novozymes), such as one with one or more,such as all, of the following substitutions: F100D, S283G, N456E, F512Y;or a strain of the genus a strain Penicillium, such as a strain of thePenicillium brasilianum disclosed in WO 2007/019442, or a strain of thegenus Trichoderma, such as a strain of Trichoderma reesei.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity such as one derivedfrom the genus Thermoascus, such as a strain of Thermoascus aurantiacus,such as the one described in WO 2005/074656 as SEQ ID NO: 2 or SEQ IDNO: 15 herein; or one derived from the genus Thielavia, such as a strainof Thielavia terrestris, such as the one described in WO 2005/074647 asSEQ ID NO: 7 and SEQ ID NO: 8; or one derived from a strain ofAspergillus, such as a strain of Aspergillus fumigatus, such as the onedescribed in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2; or onederived from a strain derived from Penicillium, such as a strain ofPenicillium emersonii, such as the one disclosed in WO 2011/041397 asSEQ ID NO: 2 or SEQ ID NO: 16 herein.

In an embodiment the cellulolytic composition comprises acellobiohydrolase I (CBH I), such as one derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus fumigatus, such asthe Cel7a CBH I disclosed in SEQ ID NO: 2 in WO 2011/057140 or SEQ IDNO: 17 herein, or a strain of the genus Trichoderma, such as a strain ofTrichoderma reesei.

In an embodiment the cellulolytic composition comprises acellobiohydrolase II (CBH II, such as one derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus fumigatus disclosedas SEQ ID NO: 18 herein; or a strain of the genus Trichoderma, such asTrichoderma reesei, or a strain of the genus Thielavia, such as a strainof Thielavia terrestris, such as cellobiohydrolase II CEL6A fromThielavia terrestris.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity and abeta-glucosidase.

In an embodiment the cellulolytic composition comprises a GH61polypeptide having cellulolytic enhancing activity, a beta-glucosidase,and a CBH I.

In an embodiment the cellulolytic composition comprises a GH61polypeptide having cellulolytic enhancing activity, a beta-glucosidase,a CBH I, and a CBH II.

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity(SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 15 herein), andAspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637).

In an embodiment the cellulolytic composition is a Trichoderma reeseicellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity(SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 15 herein) and Aspergillusfumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO:14 herein).

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition further comprising Penicilliumemersonii GH61A polypeptide having cellulolytic enhancing activitydisclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 16 herein andAspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499or SEQ ID NO: 14 herein) or a variant thereof with the followingsubstitutions F100D, S283G, N456E, F512Y (using SEQ ID NO: 14 herein fornumbering).

In a preferred embodiment the cellulolytic enzyme composition comprisingone or more of the following components:

(i) an Aspergillus fumigatus cellobiohydrolase I;

(ii) an Aspergillus fumigatus cellobiohydrolase II;

(iii) an Aspergillus fumigatus beta-glucosidase or variant thereof; and

(iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancingactivity; or homologs thereof.

In an preferred embodiment the cellulolytic enzyme composition isderived from Trichoderma reesei comprising GH61A polypeptide havingcellulolytic enhancing activity derived from a strain of Penicilliumemersonii (SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 16 herein),Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 in WO 2005/047499or SEQ ID NO: 14 herein) variant with the following substitutions:F100D, S283G, N456E, F512Y (disclosed in WO 2012/044915); Aspergillusfumigatus Cel7A CBH I disclosed as SEQ ID NO: 6 in WO2011/057140 or SEQID NO: 6 herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO:17 in WO 2011/057140 or SEQ ID NO: 18 herein.

In an embodiment the cellulolytic composition is dosed from 0.0001-3 mgEP/g DS, preferably, 0.0005-2 mg EP/g DS, preferably 0.001-1 mg/g DS,more preferably 0.005-0.5 mg EP/g DS, and even more preferably 0.01-0.1mg EP/g DS.

Proteases Present and/or Added in Saccharification and/or Fermentation

Any suitable protease may be added in saccharification and/orfermentation, such as SSF.

In a preferred embodiment the protease is a metallo protease or a serineprotease. In an embodiment the enzyme composition comprises a metalloprotease, preferably derived from a strain of the genus Thermoascus,preferably a strain of Thermoascus aurantiacus, especially Thermoascusaurantiacus CGMCC No. 0670, such as the metallo protease disclosed asthe mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or themature polypeptide of SEQ ID NO: 13 herein.

In an embodiment the protease has at least 60%, such as at least 70%,such as at least 75% identity preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, even more preferably at least 93%,most preferably at least 94%, and even most preferably at least 95%,such as even at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to the mature part of the polypeptide of SEQ ID NO: 13herein.

In an embodiment the protease is derived from a strain of Pyrococcus,such as a strain of Pyrococcus furiosus, such as the protease shown inSEQ ID NO: 1 in U.S. Pat. No. 6,358,726 or SEQ ID NO: 9 herein.

In an embodiment the protease is the mature sequence from Meripilusgiganteus protease 3 (peptidase family S53 protease) concerned inExample 2 in WO 2014/037438 (hereby incorporated by reference). In anembodiment the protease is the mature protease 3 sequence from a strainof Meripilus, in particular Meripilus giganteus shown as SEQ ID NO: 5 inWO 2014/037438 (hereby incorporated by reference) and SEQ ID NO: 19herein.

In an embodiment the protease has at least 60%, such as at least 70%,such as at least 75% identity preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, even more preferably at least 93%,most preferably at least 94%, and even most preferably at least 95%,such as even at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to the mature part of the polypeptide of SEQ ID NO: 19herein shown as amino acids 1-547.

Alpha-Amylase Present and/or Added in Saccharification and/orFermentation

Any suitable alpha-amylase, such as fungal acid alpha-amylase, may bepresent and/or added in saccharification and/or fermentation.

In a preferably embodiment the alpha-amylase is a fungal alpha-amylase,in particular one that has at least 60%, such as at least 70%, such asat least 75% identity preferably at least 80%, more preferably at least85%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to the mature part of the polypeptide of SEQ ID NO: 7. In apreferred embodiment the alpha-amylase has one or more of the followingsubstitutions: G128D, D143N, in particular G128D+D143N.

Processes of Producing a Fermentation Product from Cellulolic MaterialsUsing a Trehalase of the Invention

In an embodiment the invention relates to processes of producing afermentation product from pretreated cellulosic material, comprising:

(a) hydrolyzing said pretreated cellulosic material with a cellulolyticenzyme composition;

(b) fermenting using a fermenting organism; and

(c) optionally recovering the fermentation product,

wherein a trehalase of the invention is added and/or present inhydrolysis step (a) and/or fermentation step (b).

According to the process of the invention hydrolysis and fermentationmay be carried out separate or simultaneous. In an embodiment theprocess of the invention is carried out as separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); ordirect microbial conversion (DMC), also sometimes called consolidatedbioprocessing (CBP). SHF uses separate process steps to firstenzymatically hydrolyze the cellulosic material to fermentable sugars,e.g., glucose, cellobiose, and pentose monomers, and then ferment thefermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of thecellulosic material and the fermentation of sugars to ethanol arecombined in one step. SSCF involves the co-fermentation of multiplesugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and theenvironment: A strategic perspective on the U.S. Department of Energy'sresearch and development activities for bioethanol, Biotechnol. Prog.15: 817-827). HHF involves a separate hydrolysis step, and in addition asimultaneous saccharification and hydrolysis step, which can be carriedout in the same reactor. The steps in a HHF process can be carried outat different temperatures, i.e., high temperature enzymatic hydrolysisfollowed by SSF at a lower temperature that the fermentation strain cantolerate. DMC combines all three processes (enzyme production,hydrolysis, and fermentation) in one or more (e.g., several) steps wherethe same organism is used to produce the enzymes for conversion of thecellulosic material to fermentable sugars and to convert the fermentablesugars into a final product.

According to the invention the cellulosic material is plant materialchips, plant stem segments and/or whole plant stems. In an embodimentcellulosic material is selected from the group comprising arundo,bagasse, bamboo, corn cob, corn fiber, corn stover, miscanthus, orangepeel, rice straw, switchgrass, wheat straw. In a preferred embodimentthe source of the cellulosic material is corn stover, corn cobs, and/orwheat straw.

According to the invention any pretreatment may be used. In a preferredembodiment chemical pretreatment, physical pretreatment, or chemicalpretreatment and a physical pretreatment is used. In a preferredembodiment the cellulosic material is pretreated with an acid, such asdilute acid pretreatment. In an embodiment the cellulosic material isprepared by pretreating cellulosic material at high temperature, highpressure with an acid.

In an embodiment hydrolysis is carried out at a temperature between20-70° C., such as 30-60° C., preferably 45-55° C. at a pH in the range4-6, such as 4.5-5.5.

In an embodiment the cellulosic material is present at 1-20 (w/w) % ofTS, such as 2-10 (w/w) % TS, such as around 5 (w/w) % TS duringhydrolysis.

In an embodiment the hydrolysis is carried out for 1-20 days, preferablybetween from 5-15 days.

In an embodiment the cellulolytic enzyme composition is derived fromTrichoderma reesei, Humicola insolens or Chrysosporium lucknowense.

Cellulolytic enzyme composition: The term “cellulolytic enzymecomposition” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 21filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Examples of cellulolytic compositions can be found in the “CellulolyticEnzyme Composition present and/or added during Saccharification and/orFermentation”-section above.

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of cellulosic material is cellulose, the second most abundant ishemicellulose, and the third is pectin. The secondary cell wall,produced after the cell has stopped growing, also containspolysaccharides and is strengthened by polymeric lignin covalentlycross-linked to hemicellulose. Cellulose is a homopolymer ofanhydrocellobiose and thus a linear beta-(1-4)-D-glucan, whilehemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20 (polyoxyethylene sorbitanmonolaurate).

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). Cellobiohydrolase activity is determined according to theprocedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279;van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh andClaeyssens, 1985, FEBS Letters 187: 283-288; and Tomme et al., 1988,Eur. J. Biochem. 170: 575-581. In the present invention, the Tomme etal. method can be used to determine cellobiohydrolase activity.

Endoglucanase: The term “endoglucanase” means an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzesendohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulosederivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996,Updating the sequence-based classification of glycosyl hydrolases,Biochem. J. 316: 695-696. The enzymes in this family were originallyclassified as a glycoside hydrolase family based on measurement of veryweak endo-1,4-beta-D-glucanase activity in one family member. Thestructure and mode of action of these enzymes are non-canonical and theycannot be considered as bona fide glycosidases. However, they are keptin the CAZy classification on the basis of their capacity to enhance thebreakdown of cellulose when used in conjunction with a cellulase or amixture of cellulases.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Microbial hemicellulases. Current Opinion In Microbiology6(3): 219-228). Hemicellulases are key components in the degradation ofplant biomass. Examples of hemicellulases include, but are not limitedto, an acetylmannan esterase, an acetylxylan esterase, an arabinanase,an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates of theseenzymes, the hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature, e.g., 50C, 55° C., or 60° C., andpH, e.g., 5.0 or 5.5.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., andpH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In an aspect, a mixture ofCELLUCLAST® 1.5L (Novozymes A/S, Bagsværd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Fermenting Organism for Cellulosic Based Fermentation

The term “fermenting organism” or “fermenting microorganism” refers toany organism, including bacterial and fungal organisms, suitable for usein a desired fermentation process to produce a fermentation product. Thefermenting organism may be hexose and/or pentose fermenting organisms,or a combination thereof. Both hexose and pentose fermenting organismsare well known in the art. Suitable fermenting microorganisms are ableto ferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting organisms that can ferment hexose sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia.

In another more preferred aspect, the yeast is a Pichia stipitis. Inanother preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacillus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coil. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe processes of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products

According to the invention the term “fermentation product” can be anysubstance derived from fermentation. The fermentation product can be,without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol,ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propyleneglycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.,pentane, hexane, heptane, octane, nonane, decane, undecane, anddodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane,and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene);an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,serine, and threonine); a gas (e.g., methane, hydrogen (H₂), carbondioxide (CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g.,acetone); an organic acid (e.g., acetic acid, acetonic acid, adipicacid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formicacid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid,glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); and polyketide. The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more (e.g., several) hydroxyl moieties. In a morepreferred aspect, the alcohol is n-butanol. In another more preferredaspect, the alcohol is isobutanol. In another more preferred aspect, thealcohol is ethanol. In another more preferred aspect, the alcohol ismethanol. In another more preferred aspect, the alcohol is arabinitol.In another more preferred aspect, the alcohol is butanediol. In anothermore preferred aspect, the alcohol is ethylene glycol. In another morepreferred aspect, the alcohol is glycerin. In another more preferredaspect, the alcohol is glycerol. In another more preferred aspect, thealcohol is 1,3-propanediol. In another more preferred aspect, thealcohol is sorbitol. In another more preferred aspect, the alcohol isxylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R.,2002, The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam and Singh, 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30(2): 117-124; Ezeji et al., 2003, Production of acetone,butanol and ethanol by Clostridium beijerinckii BA101 and in siturecovery by gas stripping, World Journal of Microbiology andBiotechnology 19(6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard andMargaritis, 2004, Empirical modeling of batch fermentation kinetics forpoly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87(4): 501-515. In another preferredaspect, the fermentation product is a gas. In another more preferredaspect, the gas is methane. In another more preferred aspect, the gas isH₂. In another more preferred aspect, the gas is CO₂. In another morepreferred aspect, the gas is CO. See, for example, Kataoka, Miya, andKiriyama, 1997, Studies on hydrogen production by continuous culturesystem of hydrogen-producing anaerobic bacteria, Water Science andTechnology 36(6-7): 41-47; and Gunaseelan, 1997, Anaerobic digestion ofbiomass for methane production: A review, Biomass and Bioenergy,13(1-2): 83-114.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more (e.g., several) ketone moieties. In another morepreferred aspect, the ketone is acetone. See, for example, Qureshi andBlaschek, 2003, supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen and Lee, 1997, Membrane-mediated extractivefermentation for lactic acid production from cellulosic biomass, Appl.Biochem. Biotechnol. 63-65: 435-448.

Recovery

The fermentation product(s) are optionally recovered after fermentationusing any method known in the art including, but not limited to,chromatography, electrophoretic procedures, differential solubility,distillation, or extraction. For example, alcohol, such as ethanol, isseparated from the fermented material and purified by conventionalmethods of distillation. Ethanol with a purity of up to about 96 vol. %can be obtained, which can be used as, for example, fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

The present invention is further disclosed in the following numberedembodiments.

Embodiment 1

A polypeptide having trehalase activity, selected from the groupconsisting of:

(a) a polypeptide having at least 93% sequence identity to the maturepolypeptide of SEQ ID NO: 21 or at least 70% sequence identity to themature polypeptide of SEQ ID NO: 23;

(b) a polypeptide encoded by a polynucleotide having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 20 or the cDNA sequence thereof; or at least 80% sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 22 or the cDNAsequence thereof;

(c) a variant of the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO:23 comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions; and

(d) a fragment of the polypeptide of (a), (b), or (c), that hastrehalase activity.

Embodiment 2

The polypeptide of embodiment 1, having at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the mature polypeptide of SEQ ID NO: 21.

Embodiment 3

The polypeptide of any of embodiments 1-2, which is encoded by apolynucleotide having at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 20 or the cDNA sequence thereof.

Embodiment 4

The polypeptide of any of embodiments 1-3, comprising or consisting ofSEQ ID NO: 21 or the mature polypeptide of SEQ ID NO: 21 shown as aminoacids 19-1038 of SEQ ID NO: 21.

Embodiment 5

The polypeptide of embodiment 1, having at least 75%, at least, 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to the mature polypeptide of SEQ IDNO: 23.

Embodiment 6

The polypeptide of embodiment 1, which is encoded by a polynucleotidehaving at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 22 or the cDNA sequence thereof.

Embodiment 7

The polypeptide of embodiment 1, comprising or consisting of SEQ ID NO:23 or the mature polypeptide of SEQ ID NO: 23 shown as amino acids21-1089 of SEQ ID NO: 23.

Embodiment 8

The polypeptides of any of the preceding embodiments, having a thermaldenaturing temperature, Td, determined by TSA of at least 60° C., atleast 61° C., at least 62° C., at least 63° C., at least 64° C., atleast 65° C., at least 66° C., at least 67° C., such as at least 68° C.

Embodiment 9

A composition comprising the polypeptide of any of embodiments 1-8.

Embodiment 10

A whole broth formulation or cell culture composition comprising thepolypeptide of any of embodiments 1-8.

Embodiment 11

A polynucleotide encoding the polypeptide of any of embodiments 1-8.

Embodiment 12

A nucleic acid construct or expression vector comprising thepolynucleotide of embodiment 11 operably linked to one or moreheterologous control sequences that direct the production of thepolypeptide in an expression host.

Embodiment 13

A recombinant host cell comprising the nucleic acid construct embodiment12.

Embodiment 14

The recombinant host cell of embodiment 13, wherein the cell is a yeastcell, particularly a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell, most particularly Saccharomycescerevisiae.

Embodiment 15

A method of producing a polypeptide having trehalase activity,comprising cultivating the host cell of embodiment 13 or 14 underconditions conducive for production of the polypeptide.

Embodiment 16

The method of embodiment 15, further comprising recovering thepolypeptide.

Embodiment 17

A process of producing a fermentation product, comprising

(a) liquefying a starch-containing material with an alpha-amylase;

optionally pre-saccharifying the liquefied material before step (b);

(b) saccharifying the liquefied material;

(c) fermenting using a fermentation organism;

wherein

-   -   i) a glucoamylase;    -   ii) a trehalase of any of embodiments 1-8;    -   iii) optionally a cellulolytic enzyme composition and/or a        protease;        are present and/or added during

saccharification step (b);

fermentation step (c);

simultaneous saccharification and fermentation;

optionally presaccharification step before step (b).

Embodiment 18

The process of embodiment 17, wherein the alpha-amylase is a bacterialalpha-amylase, in particular of the genus Bacillus, such as a strain ofBacillus stearothermophilus, in particular a variant of a Bacillusstearothermophilus alpha-amylase, such as the one shown in SEQ ID NO: 3in WO 99/019467 or SEQ ID NO: 8 herein.

Embodiment 19

The process of embodiment 18, wherein the Bacillus stearothermophilusalpha-amylase or variant thereof is truncated, preferably to be from485-495 amino acids long, such as around 491 amino acids long.

Embodiment 20

The process of any of embodiments 17-19, wherein the Bacillusstearothermophilus alpha-amylase has a double deletion at positionsI181+G182, and optionally a N193F substitution, or deletion of R179+G180(using SEQ ID NO: 8 for numbering).

Embodiment 21

The process of any of embodiments 17-20, wherein the Bacillusstearothermophilus alpha-amylase has a substitution in position S242,preferably a S242A, E or Q substitution (using SEQ ID NO: 8 fornumbering).

Embodiment 22

The process of any of embodiments 17-21, wherein the Bacillusstearothermophilus alpha-amylase has a substitution in position E188,preferably E188P substitution (using SEQ ID NO: 8 for numbering).

Embodiment 23

The process of any of embodiments 17-22, wherein the alpha-amylase inliquefaction step (a) is selected from the following group of Bacillusstearothermophilus alpha-amylase variants:

I181*+G182*+N193F+E129V+K177L+R179E;

I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S

I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;

I181*+G182*+N193F+V59A+E129V+K177L+R179E+Q254S+M284V and

I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQID NO: 8 herein for numbering).

Embodiment 24

The process of any of embodiments 17-23, wherein the glucoamylase is offungal origin, preferably from a strain of Aspergillus, preferably A.niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferablyT. reesei; or a strain of Talaromyces, preferably T. emersonii, or astrain of Gloeophyllum, such as G. serpiarium or G. trabeum.

Embodiment 25

The process of any of embodiments 17-24, wherein the glucoamylase isderived from Talaromyces emersonii, such as the one shown in SEQ ID NO:4 herein.

Embodiment 26

The process of any of embodiments 17-25, wherein the glucoamylase isselected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 4 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 4 herein.

Embodiment 27

The process of any of embodiments 17-24, wherein the glucoamylase isderived from Gloeophyllum serpiarium, such as the one shown in SEQ IDNO: 5 herein.

Embodiment 28

The process embodiment 27, wherein the glucoamylase is selected from thegroup consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 5 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 5 herein.

Embodiment 29

The process of any of embodiments 17-24, wherein the glucoamylase isderived from Gloeophyllum trabeum such as the one shown in SEQ ID NO: 6herein.

Embodiment 30

The process of embodiment 29, wherein the glucoamylase present and/oradded in saccharification is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 6 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 6 herein.

Embodiment 31

The process of any of embodiments 17-30, further wherein analpha-amylase is present and/or added during saccharification step (b);fermentation step (c); simultaneous saccharification and fermentation;or the optional presaccharification step before step (b).

Embodiment 32

The process of embodiment 31, wherein the alpha-amylase is of fungal orbacterial origin.

Embodiment 33

The process of embodiment 31 or 32, wherein the alpha-amylase is derivedfrom a strain of the genus Rhizomucor, preferably a strain theRhizomucor pusillus, such as the one shown in SEQ ID NO: 3 in WO2013/006756, such as a Rhizomucor pusillus alpha-amylase hybrid havingan Aspergillus niger linker and starch-bonding domain, such as the oneshown in SEQ ID NO: 7 herein.

Embodiment 34

The process of any of embodiments 31-33, wherein the alpha-amylasepresent and/or added in saccharification and/or fermentation is selectedfrom the group consisting of:

(i) an alpha-amylase comprising the polypeptide of SEQ ID NO: 7 herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 7 herein.

Embodiment 35

The process of any of embodiments 31-34, wherein the alpha-amylase is avariant of the alpha-amylase shown in SEQ ID NO: 7 having at least oneof the following substitutions or combinations of substitutions: D165M;Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W;A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N;Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C;Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N;Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R;Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; orG128D+Y141W+D143N+K192R+P219C (using SEQ ID NO: 7 for numbering).

Embodiment 36

The process of any of embodiments 33-35, wherein the alpha-amylase isderived from a Rhizomucor pusillus, in particular with an Aspergillusniger glucoamylase linker and starch-binding domain (SBD), preferablythe one disclosed as SEQ ID NO: 7 herein, preferably having one or moreof the following substitutions: G128D, D143N, preferably G128D+D143N(using SEQ ID NO: 7 for numbering).

Embodiment 37

The process of any of embodiments 35-36, wherein the alpha-amylasevariant has at least 60% identity, such as at least 70%, preferably atleast 75% identity, preferably at least 80%, more preferably at least85%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, but lessthan 100% identity to the mature part of the polypeptide of SEQ ID NO: 7herein.

Embodiment 38

The process of any of embodiments 17-37, wherein the cellulolytic enzymecomposition is derived from Trichoderma reesei, Humicola insolens orChrysosporium lucknowense.

Embodiment 39

The process of any of embodiments 17-38, wherein the cellulolytic enzymecomposition comprises a beta-glucosidase, a cellobiohydrolase, anendoglucanase and optionally a GH61 polypeptide.

Embodiment 40

The process of any of embodiment 17-39, wherein the cellulolytic enzymecomposition comprises a beta-glucosidase, preferably one derived from astrain of the genus Aspergillus, such as Aspergillus oryzae, such as theone disclosed in WO 2002/095014 or the fusion protein havingbeta-glucosidase activity disclosed in WO 2008/057637, or Aspergillusfumigatus, such as one disclosed in SEQ ID NO: 2 in WO 2005/047499 orSEQ ID NO: 14 herein or an Aspergillus fumigatus beta-glucosidasevariant disclosed in WO 2012/044915; in particular an Aspergillusfumigatus beta-glucosidase variant with one or more, such as all, of thefollowing substitutions: F100D, S283G, N456E, F512Y; or a strain of thegenus a strain Penicillium, such as a strain of the Penicilliumbrasilianum disclosed in WO 2007/019442, or a strain of the genusTrichoderma, such as a strain of Trichoderma reesei.

Embodiment 41

The process of any of embodiments 17-40, wherein the cellulolytic enzymecomposition comprises a cellobiohydrolase I (CBH I), such as one derivedfrom a strain of the genus Aspergillus, such as a strain of Aspergillusfumigatus, such as the Cel7a CBH I disclosed in SEQ ID NO: 6 in WO2011/057140 or SEQ ID NO: 17 herein, or a strain of the genusTrichoderma, such as a strain of Trichoderma reesei.

Embodiment 42

The process of any of embodiments 17-41, wherein the cellulolytic enzymecomposition comprises a cellobiohydrolase II (CBH II, such as onederived from a strain of the genus Aspergillus, such as a strain ofAspergillus fumigatus; such as the one disclosed as SEQ ID NO: 18 hereinor a strain of the genus Trichoderma, such as Trichoderma reesei, or astrain of the genus Thielavia, such as a strain of Thielavia terrestris,such as cellobiohydrolase II CEL6A from Thielavia terrestris.

Embodiment 43

The process of any of embodiments 17-42, wherein the cellulolytic enzymecomposition further comprises a GH61 polypeptide having cellulolyticenhancing activity such as one derived from the genus Thermoascus, suchas a strain of Thermoascus aurantiacus, such as the one described in WO2005/074656 as SEQ ID NO: 2 or SEQ ID NO: 15 herein; or one derived fromthe genus Thielavia, such as a strain of Thielavia terrestris, such asthe one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; orone derived from a strain of Aspergillus, such as a strain ofAspergillus fumigatus, such as the one described in WO 2010/138754 asSEQ ID NO: 1 and SEQ ID NO: 2; or one derived from a strain derived fromPenicillium, such as a strain of Penicillium emersonii, such as the onedisclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID NO: 16 herein.

Embodiment 44

The process of any of embodiments 17-43, wherein the cellulolytic enzymecomposition is a Trichoderma reesei cellulolytic enzyme composition,further comprising Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656 or SEQID NO: 15 herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO:2 of WO 2005/047499) or SEQ ID NO: 14 herein.

Embodiment 45

The process of any of embodiments 17-44, wherein the cellulolytic enzymecomposition is a Trichoderma reesei cellulolytic enzyme compositionfurther comprising Penicillium emersonii GH61A polypeptide havingcellulolytic enhancing activity disclosed in WO 2011/041397 as SEQ IDNO: 2 or SEQ ID NO: 16 herein; and Aspergillus fumigatusbeta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 14herein) or a variant thereof with one or more, such as all, of thefollowing substitutions: F100D, S283G, N456E, F512Y.

Embodiment 46

The process of any of embodiments 17-45, wherein the cellulolytic enzymecomposition is dosed from 0.0001-3 mg EP/g DS, preferably 0.0005-2 mgEP/g DS, preferably 0.001-1 mg/g DS, more preferred from 0.005-0.5 mgEP/g DS, even more preferred 0.01-0.1 mg EP/g DS.

Embodiment 47

The process of any of embodiments 17-46, wherein the presaccharificationis carried out at a temperature from 40-75° C., such as 50-70° C.,preferably 60° C.; a pH between 4-6, preferably 5; for a period of30-360 minutes, such as from 60-420 minutes, such as around between150-180 minutes.

Embodiment 48

A process of any of embodiments 17-47, comprising the steps of:

(a) liquefying a starch-containing material with an alpha-amylase;

optionally pre-saccharifying the liquefied material before step (b);

(b) saccharifying the liquefied material;

(c) fermenting using a fermentation organism;

wherein

-   -   i) a glucoamylase;    -   ii) a trehalase of any of embodiments 1-8;        are present and/or added during

saccharification step (b);

fermentation step (c);

simultaneous saccharification and fermentation;

optionally presaccharification step before step (b).

Embodiment 49

A process of any of embodiments 17-48, comprising the steps of:

(a) liquefying a starch-containing material with an alpha-amylase;

optionally pre-saccharifying the liquefied material before step (b);

(b) saccharifying the liquefied material;

(c) fermenting using a fermentation organism;

wherein

-   -   i) a glucoamylase from Talaromyces emersonii or Gloeophyllum        serpiarium;    -   ii) a trehalase shown in any of embodiments 1-8;        are present and/or added during

saccharification step (b);

fermentation step (c);

simultaneous saccharification and fermentation;

optionally presaccharification step before step (b).

Embodiment 50

A process of any of embodiments 17-49, comprising the steps of:

(a) liquefying a starch-containing material with an alpha-amylase;

optionally pre-saccharifying the liquefied material before step (b);

(b) saccharifying the liquefied material;

(c) fermenting using a fermentation organism;

wherein

-   -   i) a glucoamylase from Talaromyces emersonii or Gloeophyllum        serpiarium;    -   ii) a trehalase shown any of embodiments 1-8;    -   iii) a cellulolytic enzyme composition derived from Trichoderma        reesei;        are present and/or added during

saccharification step (b);

fermentation step (c);

simultaneous saccharification and fermentation;

optionally presaccharification step before step (b).

Embodiment 51

The process of any of embodiments 17-50, wherein saccharification step(a) and fermentation step (b) are done separately or simultaneously.

Embodiment 52

The process of any of embodiments 17-51, wherein the fermentationproduct is recovered after fermentation.

Embodiment 53

The process of any of embodiments 17-52, wherein the starch-containingmaterial is plant material selected from the corn (maize), cobs, wheat,barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans,sweet potatoes, or a mixture thereof, preferably corn.

Embodiment 54

The process of any of embodiments 17-53, wherein the temperature inliquefaction is above the initial gelatinization temperature, inparticular in the range from 70-1000C, such as between 75-95° C., suchas between 75-90° C., preferably between 80-90° C., such as 82-88° C.,such as around 85° C.

Embodiment 55

The process of any of embodiments 17-54, wherein liquefaction step (a)is carried out at a pH in the range between 3 and 7, preferably from 4to 6, or more preferably from 4.5 to 5.5.

Embodiment 56

The process of any of embodiments 17-55, wherein the dry solid content(DS) in liquefaction lies in the range from 20-55 wt.-%, preferably25-45 wt.-%, more preferably 30-40 wt.-% or 30-45 wt-%.

Embodiment 57

The process of any of embodiments 17-56, further comprises, prior to theliquefaction step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by dry milling;

y) forming a slurry comprising the starch-containing material and water.

Embodiment 58

The process of any of embodiments 17-57, wherein a jet-cooking step iscarried out prior to liquefaction in step (a).

Embodiment 59

The process of any of embodiments 17-58, wherein the starch-containingmaterial is reduced in particle size, such by dry milling or wet millingor using particle size emulsion technology.

Embodiment 60

The process of any of embodiments 17-59, wherein the fermentation iscarried out for 30 to 150 hours, preferably 48 to 96 hours.

Embodiment 61

The process of any of embodiments 17-60, wherein the temperature duringfermentation in step (b) or simultaneous saccharification andfermentation in steps (a) and (b) is between 25° C. and 40° C.,preferably between 28° C. and 36° C., such as between 28° C. and 35° C.,such as between 28° C. and 34° C., such as around 32° C.

Embodiment 62

The process of any of embodiments 17-61, wherein further a protease ispresent during saccharification and/or fermentation.

Embodiment 63

The process of any of embodiments 17-62, wherein glucoamylase is presentand/or added in an amount of 0.001 to 10 AGU/g DS, preferably from 0.01to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS.

Embodiment 64

The process of any of embodiments 17-63, wherein the fermentationproduct is an alcohol, preferably ethanol, especially fuel ethanol,potable ethanol and/or industrial ethanol.

Embodiment 65

The process of any of embodiments 17-64, further wherein a protease ispresent and/or added during

-   -   saccharification step (b);    -   fermentation step (c);    -   simultaneous saccharification and fermentation;    -   optionally presaccharification step before step (b).

Embodiment 66

The process of any of embodiments 17-65, wherein the protease is derivedfrom Thermoascus, in particular Thermoascus aurantiacus, especiallyThermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39)shown in SEQ ID NO: 13 herein.

Embodiment 67

The process of embodiment 66, wherein the protease is the one shown inSEQ ID NO: 13 herein or a protease being at least 60%, such as at least70%, such as at least 80%, such as at least 90%, such as at least 95%,such as at least 96%, such as at least 97%, as as at least 98%, such asat least 99% identical to SEQ ID NO: 13 herein.

Embodiment 68

The process of any of embodiments 17-65, wherein the protease is derivedfrom a strain of Meripilus, in particular Meripilus giganteus, inparticular the one shown as SEQ ID NO: 19 herein.

Embodiment 69

The process of embodiment 68, wherein the protease is the one shown inSEQ ID NO: 19 herein or a protease being at least 60%, such as at least70%, such as at least 80%, such as at least 90%, such as at least 95%,such as at least 96%, such as at least 97%, as as at least 98%, such asat least 99% identical to SEQ ID NO: 19 herein.

Embodiment 70

The process of any of embodiments 17-69, wherein the fermenting organismis derived from a strain of Saccharomyces, such as Saccharomycescerevisae.

Embodiment 71

The process according to any of the embodiments 17-70, wherein the yeastfermenting organism expresses the trehalse according to embodiments 1-8.

Embodiment 72

A process of producing fermentation products from starch-containingmaterial comprising:

(i) saccharifying a starch-containing material at a temperature belowthe initial gelatinization temperature; and

(ii) fermenting using a fermentation organism;

wherein saccharification and/or fermentation is done in the presence ofthe following enzymes: glucoamylase, alpha-amylase, trehalase of any ofembodiments 1-8, and optionally a protease and/or a cellulolytic enzymecomposition.

Embodiment 73

A process of producing a fermentation product from pretreated cellulosicmaterial, comprising:

(a) hydrolyzing said pretreated cellulosic material with a cellulolyticenzyme composition;

(b) fermenting using a fermenting organism; and

(c) optionally recovering the fermentation product,

wherein a trehalase of any of embodiments 1-8 is added and/or present inhydrolysis step (a) and/or fermentation step (b).

Embodiment 74

The process of any of embodiments 17-73, wherein the trehalase is addedin an amount between 0.01-20 ug EP trehalase/g DS, such as between0.05-15 ug EP terhalase/g DS, such as between 0.5 and 10 ug EPtrehalase/g DS.

Embodiment 75

The process of any of embodiments 17-74, wherein the cellulolytic enzymecomposition is derived from Trichoderma reesei, Humicola insolens orChrysosporium lucknowense.

Embodiment 76

The process of any of embodiments 72-75, wherein the cellulolytic enzymecomposition comprising a beta-glucosidase, a cellobiohydrolase, anendoglucanase and optionally a GH61 polypeptide.

Embodiment 77

The process of any of embodiment 72-76, wherein the cellulolytic enzymecomposition comprises a beta-glucosidase, preferably one derived from astrain of the genus Aspergillus, such as Aspergillus oryzae, such as theone disclosed in WO 2002/095014 or the fusion protein havingbeta-glucosidase activity disclosed in WO 2008/057637, or Aspergillusfumigatus, such as one disclosed in SEQ ID NO: 2 in WO 2005/047499 orSEQ ID NO: 14 herein or an Aspergillus fumigatus beta-glucosidasevariant disclosed in WO 2012/044915; in particular an Aspergillusfumigatus beta-glucosidase variant with one or more, such as all, of thefollowing substitutions: F100D, S283G, N456E, F512Y; or a strain of thegenus a strain Penicillium, such as a strain of the Penicilliumbrasilianum disclosed in WO 2007/019442, or a strain of the genusTrichoderma, such as a strain of Trichoderma reesei.

Embodiment 78

The process of any of embodiments 72-77, wherein the cellulolytic enzymecomposition comprises a cellobiohydrolase I (CBH I), such as one derivedfrom a strain of the genus Aspergillus, such as a strain of Aspergillusfumigatus, such as the Cel7a CBH I disclosed in SEQ ID NO: 6 in WO2011/057140 or SEQ ID NO: 17 herein, or a strain of the genusTrichoderma, such as a strain of Trichoderma reesei.

Embodiment 79

The process of any of embodiments 72-78, wherein the cellulolytic enzymecomposition comprises a cellobiohydrolase II (CBH II, such as onederived from a strain of the genus Aspergillus, such as a strain ofAspergillus fumigatus; such as the one disclosed as SEQ ID NO: 18 hereinor a strain of the genus Trichoderma, such as Trichoderma reesei, or astrain of the genus Thielavia, such as a strain of Thielavia terrestris,such as cellobiohydrolase II CEL6A from Thielavia terrestris.

Embodiment 80

The process of any of embodiments 72-79, wherein the cellulolytic enzymecomposition further comprises a GH61 polypeptide having cellulolyticenhancing activity such as one derived from the genus Thermoascus, suchas a strain of Thermoascus aurantiacus, such as the one described in WO2005/074656 as SEQ ID NO: 2 or SEQ ID NO: 15 herein; or one derived fromthe genus Thielavia, such as a strain of Thielavia terrestris, such asthe one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; orone derived from a strain of Aspergillus, such as a strain ofAspergillus fumigatus, such as the one described in WO 2010/138754 asSEQ ID NO: 1 and SEQ ID NO: 2; or one derived from a strain derived fromPenicillium, such as a strain of Penicillium emersonii, such as the onedisclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID NO: 16 herein.

Embodiment 81

The process of any of embodiments 72-80, wherein the cellulolytic enzymecomposition is a Trichoderma reesei cellulolytic enzyme composition,further comprising Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656 or SEQID NO: 15 herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO:2 of WO 2005/047499) or SEQ ID NO: 14 herein.

Embodiment 82

The process of any of embodiments 72-81, wherein the cellulolytic enzymecomposition is a Trichoderma reesei cellulolytic enzyme compositionfurther comprising Penicillium emersonii GH61A polypeptide havingcellulolytic enhancing activity disclosed in WO 2011/041397 as SEQ IDNO: 2 or SEQ ID NO: 16 herein; and Aspergillus fumigatusbeta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 14herein) or a variant thereof with one or more, such as all, of thefollowing substitutions: F100D, S283G, N456E, F512Y.

Embodiment 83

The process of any of embodiments 72-82, wherein the cellulolytic enzymecomposition is dosed from 0.0001-3 mg EP/g DS, preferably 0.0005-2 mgEP/g DS, preferably 0.001-1 mg/g DS, more preferred from 0.005-0.5 mgEP/g DS, even more preferred 0.01-0.1 mg EP/g DS.

The present invention is further described by the following examples.

EXAMPLES

Materials & Methods

Enzymes and Yeast Used:

Alpha-Amylase BE369 (AA369):

Bacillus stearothermophilus alpha-amylase disclosed herein as SEQ ID NO:8, and further having the mutations:I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to491 amino acids (using SEQ ID NO: 8 for numbering).

Protease PfuS shown in SEQ ID NO: 9 herein.

Glucoamylase X:

Blend comprising Talaromyces emersonii glucoamylase disclosed as SEQ IDNO: 34 in WO99/28448 (SEQ ID NO: 4 herein), Trametes cingulataglucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289 (SEQ ID NO: 11herein), and Rhizomucor pusillus alpha-amylase with Aspergillus nigerglucoamylase linker and starch binding domain (SBD) disclosed in SEQ IDNO: 7 herein having the following substitutions G128D+D143N using SEQ IDNO: 7 for numbering (activity ratio in AGU:AGU:FAU-F is about 29:8:1).

Yeast: Ethanol Red®

Trehalase Assay:

Principle:

${\left. {{Trehalose} + {H_{2}O\mspace{11mu}\underset{\_}{Trehalase}}} \right\rangle\mspace{14mu} 2\mspace{20mu}{Glucose}}\mspace{11mu}$T=37° C., pH=5.7, A340 nm, Light path=1 cmSpectrophotometric Stop Rate DeterminationUnit Definition:

One unit will convert 1.0 mmole of trehalose to 2.0 mmoles of glucoseper minute at pH 5.7 at 37° C. (liberated glucose determined at pH 7.5).

(See Dahlqvist, A. (1968) Analytical Biochemistry 22, 99-107)

Trehalase Assay Used in Example 3.

Ten microliters of sample was mixed with 190 μl of substrate solution(1% trehalose in 50 mM sodium acetate, pH 4.3) and incubate at 32° C.for 1 hour. 10 μl of the solution was then taken out and 200 μl ofglucose CII test WAKO was added. A505 was measured after 15min-incubation at room temperature.

Strains

An improved Aspergillus oryzae host/vector system comparable to the onedescribed in example 5 disclosed in WO 2016026938A1 was constructed. Theimprovement was made to reduce the size of the transforming DNA bymoving the FLPase expression cassette located on PART-II of the plasmidpDAu724 (see page 34, FIG. 7 and SEQ ID NO:30 in WO 2016026938A1) to theintegration locus amy2 in the genome of the host strain. The cloning ofthe FLPase expression cassette into pDAu703 (WO 2016026938A1 page 32 andFIG. 6 and SEQ ID:29) was done by amplification of the FLPase expressioncassette from pDAu724 and cloning in between FRT-F3 and the amdSselection marker of pDAu703 to give the plasmid pDAu770. The sameprotocol as described in WO 2016026938A1 page 33 was used to transformthe linearized plasmid pDAu770 into protoplasts of A. oryzae strainJa11338 (disclosed in WO12160097A1). Transformants were selected on AmdSselection plates to obtain strain DAu785. The resulting recombinant hoststrain DAu785 has a modified amy2 locus comparable to the one in DAU716(WO 2016026938A1) with the addition of the FLPase expression cassette.The host strain DAu785 is constitutively expressing the FLPase sitespecific recombinase allowing the integration at the FRT sites of thetransforming DNA in this case the PCR fragments obtained by OverlapExtension PCR reaction described below. This strain was used forheterologous expression of the trehalase polypeptides SEQ ID NO: 21, andSEQ ID NO: 23.

Talaromyces funiculosus NRRL 1035 was kindly obtained in February 1992from Pr. Jens Frisvad (Denmark Technical University, Department ofBiotechnology and Biomedicine). T. funiculosus was originally isolatedby George Smith in England in 1936. The strain was inoculated onto a PDAplate and incubated for 8 days at 26° C. in the darkness. Severalmycelia-PDA plugs were inoculated into 500 ml shake flasks containing100 ml of YPG medium. The shake flasks were incubated for 5 days at 26°C. with shaking at 100 rpm for production of biomass.

Talaromyces leycettanus reference CBS 398.68 (isolated in 1968 inEngland) was purchased from CBS-KNAW Fungal Biodiversity Centre,Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands and inoculated onto aPDA plate and incubated for 8 days at 26° C. in the darkness. Severalmycelia-PDA plugs were inoculated into 500 ml shake flasks containing100 ml of YPG medium. The flasks were incubated for 5 days at 26° C.with shaking at 100 rpm for production of biomass.

Media and Solutions

PDA plates were composed of 39 g Potato Dextrose Agar (ref. 70139)(Sigma-Aldrich, Munich, Germany) and deionized water to 1000 ml. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

LB-Bouillon was composed of 25 g of LB Bouillon (ref. L3152)(Sigma-Aldrich, Munich, Germany) and deionised water to 1000 ml. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

Ampicillin LB-agar was composed of 37 g LB agar (ref. L3027)(Sigma-Aldrich, Munich, Germany), 5 g soluble starch, 0.01 M K2PO4,0.04% glucose, and deionised water to 1000 ml. The medium was sterilizedby autoclaving at 15 psi for 15 minutes (Bacteriological AnalyticalManual, 8th Edition, Revision A, 1998). Medium was cooled to 50° C. and50 mM ampicillin was added.

COVE-N-agar plates were composed of 218 g of sorbitol, 25 g of agarpowder, 50 ml of COVE salt solution, and deionized water to 1000 ml. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 50° C. and 10 mM acetamide, and Triton X-100 (50μl/500 ml) were added.

Sucrose Agar 10 mM NaNO3 was composed of 342 g sucrose, 20 g agarpowder, 20 ml COVE salt solution, and deionized water to 1000 ml. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 50° C. and 10 mM NaNO3, and Triton X-100 (50 μl/500ml) were added.

COVE salt solution was composed of 26 g of MgSO4.7H2O, 26 g of KCL, 76 gof KH2PO4, 50 ml of COVE trace metal solution, and deionized water to1000 ml. Solution was sterile filtered.

COVE trace metal solution was composed of 0.04 g of Na2B4O7.10H2O, 0.4 gof CuSO4.5H2O, 1.2 g of FeSO4.7H2O, 0.7 g of MnSO4.H2O, 0.8 g ofNa2MoO4.2H2O, 10 g of ZnSO4.7H2O, and deionized water to 1000 ml.Solution was sterile filtered.

DAP4C-1 medium was composed of 0.5 g yeast extract, 10 g maltose, 20 gdextrose, 11 g MgSO4.7H2O, 1 g KH2PO4, 2 g C6H8O7.H2O, 5.2 g K3PO4.H2O,1 ml Dowfax 63N10 (antifoaming agent), 2.5 g calcium carbonate,supplemented with 0.5 ml KU6 trace metal solution, and deionised waterto 1000 ml. The medium was sterilized by autoclaving at 15 psi for 15minutes (Bacteriological Analytical Manual, 8th Edition, Revision A,1998). Before use, 3.5 ml of sterile 50% (NH4)2HPO4 and 5 ml of sterile20% lactic acid were added per 150 ml of DAP4C-1 medium.

MDU-2 medium was composed of 45 g maltose, 1 g MgSO4.7H2O, 1 g NaCl, 2 gK2SO4, 12 g KH2PO4, 0.5 ml KU6 trace metal, 0.1 ml Dowfax63N10(antifoaming agent), and deionised water to 1000 ml. pH was adjusted topH5 and the medium was sterilized by autoclaving at 15 psi for 15minutes (Bacteriological Analytical Manual, 8th Edition, Revision A,1998). 15 ml 50% sterile filtered urea was added after autoclaving.

YP2% glucose was composed of 10 g yeast extract, 20 g Bacto peptone, 20g dextrose, and deionized water to 1000 ml. The medium was sterilized byautoclaving at 15 psi for 15 minutes (Bacteriological Analytical Manual,8th Edition, Revision A, 1998).

KU6 trace metal solution was composed of 6.8 g ZnCl2, 2.5 g CuSO4.5H2O,0.13 g NiCl2, 13.9 g FeSO4.7H2O, 8.45 g MnSO4.H2O, 3 g C6H8O7.H2O, anddeionised water to 1000 ml. Solution was sterile filtered.

Example 1: Cloning of Trehalase Sequence

Talaromyces DNA sequences were PCR amplified from gDNA from Talaromycesfuniculosus and Talaromyces leycettanus and cloned by overlap-extensionPCR. pDAu724 plasmid (see Strain section above) was used as DNA templateto amplify two PCR products (F1 and F3) in reactions composed of 10 μlof KAPA polymerase buffer 5×, 1 μl 10 mM KAPA PCR Nucleotide Mix, 1 μlof 10 μM of the appropriate forward primers (SEQ ID NO: 24 for F1 andSEQ ID NO: 26 for F3), 1 μl of 10 μM of the appropriate reverse primers(SEQ ID NO: 25 for F1 and SEQ ID NO: 27 for F3), 1 to 10 ng of pDAu724plasmid, 1 μl of KAPA Biosystems polymerase KK2502 (1 unit) andPCR-grade water up to 50 μL. PCR amplification reactions were carriedout on a DYAD® Dual-Block Thermal Cycler (MJ Research Inc., Waltham,Mass., USA) programmed for 2 min. at 98° C. and followed by 35 cycles of10 sec. at 98° C. and 2 min. at 72° C. and one final cycle of 10 min. at72° C. Five μl of the PCR reaction were analysed by 1% agarose gelelectrophoresis using TAE buffer where DNA bands of the appropriate sizewere observed. The remaining PCR reactions were purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

Overlap Extension PCR reactions for cloning trehalase genes amplifiedfrom Talaromyces funiculosus, and Talaromyces leycettanus respectivelywere composed of 10 μl KAPA polymerase buffer (5×), 1 μl 10 mM KAPA PCRNucleotide Mix, 50 ng of PCR fragment F1 and equimolar amounts of PCRfragment F3 and trehalase genes encoding for SEQ ID NO: 21, or SEQ IDNO: 23, 1 μl KAPA Biosystems polymerase KK2502 (1 unit) and PCR-gradewater up to 48 μL. Reactions were incubated on a DYAD® Dual-BlockThermal Cycler (MJ Research Inc., Waltham, Mass., USA) using a programcomposed of 2 min. at 98° C., followed by 5 cycles each composed of 10sec. at 98° C., 30 sec. at 68° C., and 5 min. at 72° C. and completed bya final extension of 8 min. at 72° C. During the Overlap Extention PCRreactions, annealing between fragment F1 and trehalase genes encodingfor SEQ ID NO: 21, and SEQ ID NO: 23 was ensured by overlap sequence SEQID NO: 28 included in the forward cloning primers (SEQ ID NO: 31 and SEQID NO: 30). Annealing between fragment F3 and the trehalase genesencoding for SEQ ID NO: 21, SEQ ID NO: 23 was ensured by the overlapsequence SEQ ID NO: 29 included in the reverse cloning primers (SEQ IDNO: 33 and SEQ ID NO: 32). One μl of 10 μM primer SEQ ID NO: 24 and 1 μlof 10 μM primer SEQ ID NO: 27 were added to the Overlap Extention PCRreactions after the five initial cycles and the reactions were incubateda second time on a DYAD® Dual-Block Thermal Cycler (MJ Research Inc.,Waltham, Mass., USA) using a program composed of 2 min. at 98° C.;followed by 25 cycles each composed of 10 sec. at 98° C., and 4 min. at72° C. and completed by a final extension of 10 min. at 72° C. The PCRreactions resulted in two products: SEQ ID NO: 20 and SEQ ID NO: 22.Five μl of the PCR reactions were analysed by 1% agarose gelelectrophoresis using TAE buffer where an DNA bands of the appropriatesize were observed. The remaining PCR reactions were up-concentrated to20 μl by heating the tubes at 60° C. Ten μl of those reactions were usedfor Aspergillus oryzae DAu785 protoplasts transformation.

Example 2: Heterologous Expression of Trehalases

Protoplasts of Aspergillus oryzae DAu785 strain were prepared accordingto WO 95/002043. 100 μl of A. oryzae protoplasts were mixed with 10 μlof up-concentrated Overlap Extention PCR encoding for Talaromycesfuniculosus, and Talaromyces leycettanus trehalases polypeptide (aminoacids 19-1038 of SEQ ID NO: 21 and amino acids 21-1089 of SEQ ID NO:23), and 270 μl of 60% PEG 4000 (Applichem, Darmstadt, Germany)(polyethylene glycol, molecular weight 4,000), 10 mM CaCl2), and 10 mMTris-HCl pH 7.5 and gently mixed. The mixture was incubated at 37° C.for 30 minutes and the protoplasts were spread onto Sucrose Agar platescontaining 10 mM NaNO3. After incubation for 4-7 days at 37° C., sporesof eight colonies were inoculated into MDU-2 medium in 96-well X50microtiter plate PS from ThermoFisher (Life Technologies Europe BV,Naerum, Denmark) and covered with semi-permeable tape. After 4 days ofstatic incubation at 30° C., the culture broths were analysed by sodiumdodecyl sulfate polyacrylamide gel electrophoresis to identify coloniesproducing the highest amount of trehalase polypeptides. Spores of thebest transformant were spread onto Sucrose Agar plates containing 0.01%TRITON® X-100 and 10 mM NaNO3 to isolate single colonies. The spreadingwas repeated twice.

Example 3: Characterization of Trehalases of the Invention

Trehalases Organism GH Ms-trehalase Myceliophthora sepedonium 37Tr-trehalase Trichoderma reesei 65 An-trehalase Aspergillus niger 65Tl-trehalase Talaromyces leycettanus 65 Tf-trehalase Talaromycesfuniculosus 65Purification

Purification of trehalase enzymes were carried out by two steps,desalting column and cation exchange chromatography column. Finally, thesample was dialyzed against 10 L of 20 mM sodium acetate buffer (pH 4.0)using 12 k-14 k MWCO (molecular weight-cutoff) dialysis membrane andthen concentrated using 30 k MWCO centrifugal filter unit.

Thermostability Determination (TSA)

Purified enzyme was diluted to 0.5 mg/ml with 50 mM sodium acetatebuffer (pH 4.5) and mixed with equal volume of SYPRO Orange (Invitrogen)diluted with Milli-Q water. Eighteen microliters of mixture solution wastransfer to LightCycler 480 Multiwell Plate 384 (Roche Diagnostics) andthe plate was sealed.

Equipment Parameters of TSA:

Apparatus: LightCycler 480 Real-Time PCR System (Roche Applied Science)

Scan rate: 0.02° C./sec

Scan range: 37-96° C.

Integration time: 1.0 sec

Excitation wave length 465 nm

Emission wave length 580 nm

The obtained fluorescence signal was normalized into a range of 0 and 1.The denaturing temperature (Td) was defined as the temperature where thenormalized value is closest to 0.5. The temperature where the maximumsignal intensity (normalized value is 1) was defined as Td2 and it usedfor an index of thermostability of M-tre PE variants.

Trehalase Assay

Ten microliters of sample was mixed with 190 μl of substrate solution(1% trehalose in 50 mM sodium acetate, pH 4.3) and incubate at 32° C.for 1 hour. 10 μl of the solution was then taken out and 200 μl ofglucose CII test WAKO was added. A505 was measured after 15min-incubation at room temperature.

SF Cultivation for Protease Cocktail Preparation

Aspergillus niger strains used to prepare protease cocktail isderivatives of NN059095, which was isolated by Novozymes and geneticallymodified to disrupt expression of amyloglycosidase activities fromAspergillus niger NN049184 isolated from soil.

Spores of Aspergillus niger strains were inoculated in 100 ml MLC mediaand cultivated at 30° C. for 2 days. 10 ml of MLC was inoculated to 100ml of MU-1 medium and cultivated at 30° C. for 7 days. The supernatantwas obtained by centrifugation.

MLC is composed of 40 g glucose, 50 g soybean powder, 4 g citric acid(pH 5) and water to 1 liter.

MU-1-glu is composed of 260 g glucose, 3 g MgSO₄-7H₂O, 5 g KH₂PO₄, 6 gK₂SO₄, 0.5 ml of trace metal solution and 2 g urea (pH 4.5) and water to1 liter.

Trace metal solution is composed of 6.8 g ZnCl₂-7H₂O, 2.5 g CuSO₄.5H₂O,0.24 g NiCl₂′6H₂O, 13.9 g FeSO₄.7H₂O, 13.5 g MnSO₄—H₂O and 3 g citricacid and water to 1 liter.

Protease Stability Assay

Purified enzyme was diluted to 2 mg/ml with 50 mM sodium acetate bufferpH 4.0 and 100 μl of the sample was mixed with 100 μl of preparedprotease cocktail. The solution was then incubated at −20, 4, 30 and 40°C. for 3 days and the residual activity was measured by trehalase assay.

Results

Protease stability* Trehalases Td [° C.] 4° C. 30° C. 40° C.Ms-trehalase 56.9  90%  42%  15% SEQ ID NO: 1 Tr-trehalase 57.9 100%100% 100% SEQ ID NO: 2 An-trehalase 53.1 100%  97%  68% SEQ ID NO: 3Tl-trehalase 69.0 100% 100% 100% SEQ ID NO: 23 Tf-trehalase 65.2 100%100% 100% SEQ ID NO: 21 *Residual activity after 3 days-incubation in anadmixture with protease cocktail (−20° C. as 100%)

Example 4: Thermo-Stability of the Trehalases According to the InventionCompared to Two Prior Art Trehalases

Thermo-stability of the trehalases of the invention, disclosed herein asSEQ ID NO: 21 and SEQ ID NO: 23, where compared to two prior arttrehalses; one from Talaromyces cellulolyticus (SEQ ID NO: 34) (Fujii T,Hoshino T, Inoue H, Yano S. 2014. Taxonomic revision of thecellulose-degrading fungus Acremonium cellulolyticus nomen nudum toTalaromyces based on phylogenetic analysis. FEMS Microbiology Letters.351: 32-41) and one from Talaromyces verruculosus (SEQ ID NO: 35)(published in 2015 as part of a genome sequence on the NCBI website asassembly GCA 001305275.1; polypeptide identified as EFP5BRM8N). Thethermo-stability was measured as denaturing temperature using a ThermalShift Assay (TSA).

Samples

Trehalase Organism EXP13116 (SEQ ID NO: 34) Talaromyces celluloyticusEXP13117 (SEQ ID NO: 35) Talaromyces verruculosus Tl-trehalase (SEQ IDNO: 23) Talaromyces leycettanus Tf-trehalase (SEQ ID NO: 21) TalaromycesfuniculosusStrain Cultivation

Agar pieces of a strain cultivated onto COVE N-gly agar plate for 1 weekat 30° C. were inoculated to 100 ml of MS9 in a 500 ml shaking flask andit was cultivated at 30° C. for 1 day with shaking at 220 rpm. Three mlof the seed culture was transferred to 100 ml of MDU-2BP-FuPE in 500 mlshaking flask and it was cultivated at 30° C. for 3 days with shaking at220 rpm. The culture supernatant was filtrated with 0.2 μm celluloseacetate filter. Media components are described below.

COVE Nqly Aqar

218 g/L Sorbitol, 10 g/L Glycerol, 2.02 g/L Potassium Nitrate, 50 ml/Lsalt solution for COVE, pH 5.3

Salt Solution for COVE

26 g/L Potassium Chloride, 26 g/L Magnesium Sulfate Heptahydrate, 76 g/LPotassium Dihydrogenphosphate, 50 ml/L Trace metal solution for COVE

Trace Metal Solution for COVE

0.04 g/L Sodium Tetraborate Decahydrate, 0.4 g/L Copper (II) SulfatePentahydrate, 1.2 g/L Iron(II) Sulfate Heptahydrate, 1 g/L Manganese(II)Sulfate Pentahydrate, 0.8 g/L Sodium molybdate dihydrate, 10 g/L ZincSulfate Heptahydrate

MS9

30 g/L Soybean powder, 20 g/L Glycerol

MDU-2BP FuPE

45 g/L Maltodextrin, 7 g/L Yeast extract, 1 g/L Magnesium SulfateHeptahydrate, 1 g/L Sodium Chloride, 2 g/L Potassium Sulfate, 0.75 g/LAmmonium Chloride, 12 g/L Potassium Dihydrogenphosphate, 0.5 ml/L Tracemetal solution for AMG (MU-1)

Trace Metal Solution for AMG (MU-1)

1.39% Iron (II) Sulfate Heptahydrate, 1.356% Manganese(II) SulfatePentahydrate, 0.68% Zinc Chloride, 0.25% Copper(II) SulfatePentahydrate, 0.024 g/L Nickel (II) Chloride Hexahydrate, 0.3% Citricacid

Purification

Purification of WT trehalases were carried out by two steps, desaltingcolumn and cation exchange chromatography column. Finally, the samplewas dialyzed against 10 L of 20 mM sodium acetate buffer (pH 4.0) using12 k-14 k MWCO dialysis membrane and then concentrated using 30 k MWCOcentrifugal filter unit.

Thermostability Determination (TSA)

Purified enzyme was diluted to 0.5 mg/ml with 50 mM sodium acetatebuffer (pH 4.5) and mixed with equal volume of SYPRO Orange (Invitrogen)diluted with Milli-Q water. Eighteen microliters of mixture solution wastransfer to LightCycler 480 Multiwell Plate 384 (Roche Diagnostics) andthe plate was sealed.

Equipment Parameters of TSA

Apparatus: LightCycler 480 Real-Time PCR System (Roche Applied Science)

Scan rate: 0.02° C./sec

Scan range: 37-96° C.

Integration time: 1.0 sec

Excitation wave length 465 nm

Emission wave length 580 nm

The obtained fluorescence signal was normalized into a range of 0 and 1.The denaturing temperature (Td) was defined as the temperature where thenormalized value is closest to 0.5. Td are listed in TABLE 1.

TABLE 1 List of Td Trehalase Td [° C.] EXP13116 51.7 EXP13117 47.1Tl-trehalase 67.5 Tf-trehalase 64.7

Example 5: Use of Tf-Trehalase of the Invention in a Process forProducing Ethanol

Enzymes and Yeast Used:

Alpha-Amylase BE369 (AA369):

Bacillus stearothermophilus alpha-amylase disclosed herein as SEQ ID NO:8, and further having the mutations:I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to491 amino acids (using SEQ ID NO: 8 for numbering).

Protease PfuS shown in SEQ ID NO: 9 herein.

Glucoamylase X:

Blend comprising Talaromyces emersonii glucoamylase disclosed as SEQ IDNO: 34 in WO99/28448 (SEQ ID NO: 4 herein), Trametes cingulataglucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289 (SEQ ID NO: 11herein), and Rhizomucor pusillus alpha-amylase with Aspergillusnigerglucoamylase linker and starch binding domain (SBD) disclosed inSEQ ID NO: 7 herein having the following substitutions G128D+D143N usingSEQ ID NO: 7 for numbering (activity ratio in AGU:AGU:FAU-F is about29:8:1).

Yeast: Ethanol Red®

The ability of trehalase to reduce trehalose was evaluated by running asaccharification and fermentation (SSF) using either Glucoamylase blendX alone or with the addition of either Ms or Tf trehalase. The Mstrehalase dose was set at 1 μg enzyme protein (EP) per gram of drysolids (DS) while three Tf trehalose doses were used for comparison(0.6, 0.7 and 0.8 ug EP/g DS). Glucoamylase blend X were dosed at 0.6AGU/g DS and in according to the following calculation. Two plant mashes(corn starch liquefied using alpha-amylase BE369 or BE369 and proteasePfuS) were used. Mash 1 represented BE369+PfuS and Mash 2 representedBE369 mash. For Mash 1 BE369 and PfuS were dosed at 2.3 μg EP/g DS and2.6 μg EP/g DS, respectively. For Mash 2 BE369 dose was 2.81 μg EP/g DS.The urea dose for Mash 1 and Mash 2 were 400 ppm and 1000 ppm,respectively.

${{{Enz}.\mspace{11mu}{dose}}\mspace{14mu}({uL})} = \frac{\begin{matrix}{{Total}\mspace{14mu}{GA}\mspace{14mu}{{dose}\left( \frac{AGU}{g\mspace{14mu}{DS}} \right)} \times {Mass}\mspace{14mu}{Weight}\mspace{14mu}(g) \times} \\{{Dry}\mspace{14mu}{solid}\mspace{14mu}{content}\mspace{14mu}\left( {\%\mspace{14mu}{DS}} \right) \times 1000}\end{matrix}}{{Stock}\mspace{14mu}{enzyme}\mspace{14mu}{{conc}.\mspace{11mu}\left( \frac{AGU}{mL} \right)}}$

TABLE 2 Enzyme treatment in SSF of two industrial plant mashes TrehalaseDose (ug Glucoamylase Trehalase treatment EP/g DS) Blend X None 0 BlendX Ms-trehalase 1 Blend X Tf-trehalase 0.6 Blend X Tf-trehalase 0.7 BlendX Tf-trehalase 0.8

The blends were evaluated in an SSF using two liquefied industrial plantcorn mashes Mash 1 and Mash 2. The mashes were supplemented with 3 ppmpenicillin. The amount of urea added to Mash 1 and Mash 2 were 400 ppmand 1000 ppm, respectively. The pH of both plant mashes was adjusted topH 5.1 using 40% H₂SO₄ prior to dispensing mash into flasks.Approximately 60 g (55±0.03) of mash was added into each 125 mL and theywere run in duplicates. Corning Disposable Erlenmeyer flask that had a0.048″ hole on the cap for venting. Each flask was dosed with enzymesaccording to Table 2 and 1.2 mL of rehydrated Ethanol Red yeast. Ethanolred was rehydrated by resuspending 5.5 g yeast with 100 mL tap water andincubated at 32° C. for 30-40 min. Flasks were incubated for the totalof 52 hr at 32° C. while shaking at 120 rpm. Samples were collected at52 hrs. Collected samples were prepared for HPLC by removingapproximately 4 grams of fermentation sample and mixed it with 40 uL of40% H₂SO₄. The mixture was centrifuged for 10 minutes at 3500×g, and thesupernatant was filtered through 0.2 μM Whatman nylon filter. Filteredsamples were analyzed on an Agilent HPLC 1100/1200 series withChemstation software. Samples were separated on Bio-Rad HPX-87H IonExclusion column (300 mm×7.8 mm) with a cation H guard cartridge. Themobile phase, 5 mM H₂SO₄, was run at 0.8 ml/min at 65° C. and the RIdetector temperature was set at 55° C. The method quantifies severalanalytes using calibration standards (4 point calibration with forcedthrough zero) for dextrins (DP4+), maltotriose (DP3), maltose (DP2),glucose (DP1), fructose, acetic acid, lactic acid, glycerol and ethanol.HPLC results on ethanol, DP2 (maltose) are shown in Table 3. DP2 wasanalyzed since this sugar also contain the trehalose. In addition to theHPLC analysis, filtered samples of only Mash 1 were also analyzed on aDionex ICS-3000 ion chromatography (IC) using Chromeleon 5 software toseparate trehalose from other DP2 sugars (such as isomaltose, maltoseand cellobiose). Samples were separated using the Carbopack PA1 column.The mobile phase were water, 0.2 M NaOH, 1 M sodium acetate and theflowrate was 1 mL/min and both the column and detector (PAD detectorwith disposable gold electrode) were at 30° C. The IC was run for 65minutes and the trehalose level were reported in Table 4. Tf-trehalasehas slightly better performance than Ms-trehalase since 0.6-0.8 ug EP ofTf had similar level of DP2 following SSF for 52 hrs. Without anytrehalase addition, there were significant amount of DP2 left at the endof fermentation. The ethanol levels were compared between Ms andTf-trehalase treatment in both plant mashes and there was no statisticalsignificance between them. Therefore, Tf-trehalase, at 20% to 40% lowerprotein dose relative to Ms-trehalase, has similar applicationperformance as Ms-trehalase in SSF using two different types of plantmash.

TABLE 3 Ethanol, and DP2 levels following SSF in two plant mashes for 52Hrs Trehalase Average ethanol level Average DP2 Gluco- dose (μg (% w/v)level (% w/v) amylase Trehalase EP/g DS) Mash 1 Mash 2 Mash 1 Mash 2Blend X None 13.517 13.0545 0.2405 0.1785 Ms-trehalase 1 13.590 13.0800.141 0.110 Tf-trehalase 0.6 13.622 13.101 0.140 0.109 Tf-trehalase 0.713.608 13.122 0.139 0.106 Tf-trehalase 0.8 13.612 13.151 0.141 0.111

TABLE 4 Trehalose levels following SSF in two plant mashes for 52 HrsTrehalase Average trehalose dose (μg level (% w/v) GlucoamylaseTrehalase EP/g DS) Mash 1 Mash 2 Blend X None 0.0661 n/a Ms-trehalase 10.0134 n/a Tf-trehalase 0.6 0.0150 n/a Tf-trehalase 0.7 0.0147 n/aTf-trehalase 0.8 0.0151 n/a

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. A process of producing a fermentationproduct, comprising (a) liquefying a starch-containing material with analpha-amylase; optionally presaccharifying the liquefied material beforestep (b); (b) saccharifying the liquefied material; (c) fermenting usinga fermentation microorganism; wherein i) a glucoamylase; and ii) apolypeptide having trehalase activity selected from the group consistingof a polypeptide having at least 93% sequence identity to the maturepolypeptide of SEQ ID NO: 21 and a polypeptide encoded by apolynucleotide having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 20 or the cDNA sequencethereof; and iii) optionally a cellulolytic enzyme composition, aprotease, or both a cellulolytic enzyme composition and a protease arepresent or added during the saccharification step (b); the fermentationstep (c); or the optionally presaccharification step before step (b). 2.The process of claim 1, wherein an alpha-amylase is present and/or addedduring saccharification step (b); fermentation step (c); or the optionalpresaccharification step before step (b).
 3. The process of claim 1,wherein the alpha-amylase is the one disclosed as SEQ ID NO: 7, havingone or more of the following substitutions: G128D, D143N, or G128D+D143N(using SEQ ID NO: 7 for numbering), and wherein the alpha-amylasevariant has at least 80%, but less than 100% identity to the mature partof the polypeptide of SEQ ID NO:
 7. 4. The process of claim 1, whereinthe fermenting microorganism is derived from a strain of Saccharomyces.5. The process according to claim 4, wherein the yeast fermentingorganism expresses the polypeptide having trehalase activity.
 6. Aprocess of producing fermentation products from starch-containingmaterial comprising: (i) saccharifying a starch-containing material at atemperature below the initial gelatinization temperature; and (ii)fermenting using a fermentation microorganism; wherein saccharification,fermentation, or saccharification and fermentation is done in thepresence of the following enzymes: a. glucoamylase; b. alpha-amylase;and c. a polypeptide having trehalase activity selected from the groupconsisting of a polypeptide having at least 93% sequence identity to themature polypeptide of SEQ ID NO: 21 and a polypeptide encoded by apolynucleotide having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 20 or the cDNA sequencethereof; and d. optionally a protease, a cellulolytic enzymecomposition, or a protease and a cellulolytic composition.
 7. Theprocess of claim 1, wherein steps (b) and (c) are performedsimultaneously in a simultaneous saccharification and fermentation. 8.The process of claim 7, wherein an alpha-amylase is present or addedduring the simultaneous saccharification and fermentation.
 9. Theprocess of claim 7, wherein the cellulolytic enzyme composition ispresent or added during the simultaneous saccharification andfermentation.
 10. The process of claim 7, wherein the protease ispresent or added during the simultaneous saccharification andfermentation.